Therapeutic compounds for ovarian cancer

ABSTRACT

The present invention provides synthetic compounds, antibodies that recognize and bind to these compounds, polynucleotides that encode these compounds, and immune effector cells raised in response to presentation of these epitopes. The invention further provides methods for inducing an immune response and administering immunotherapy to a subject by delivering the compositions of the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. §119 (e) to U.S.Provisional Application Serial Nos. 60/209,388 and 60/257,007, filed May31, 2000 and Dec. 20, 2000, respectively. The contents of theseapplications are hereby incorporated by reference into the presentdisclosure.

TECHNICAL FIELD

[0002] The invention relates to the field of therapeutic compoundsuseful against human ovarian cancer.

BACKGROUND OF THE INVENTION

[0003] The recognition of antigenic epitopes presented by molecules ofthe Major Histocompatibility Complex (MHC) plays a central role in theestablishment, maintenance and execution of mammalian immune responses.T cell surveillance and recognition of peptide antigens presented bycell surface MHC molecules expressed by somatic cells and antigenpresenting leukocytes functions to control invasion by infectiousorganisms such as viruses, bacteria, and parasites. In addition it hasnow been demonstrated that antigen-specific cytotoxic T lymphocytes(CTLs) can recognize certain cancer cell antigens and attack cellsexpressing these antigens. This T cell activity provides a basis fordeveloping novel strategies for anti-cancer vaccines. Furthermore,inappropriate T cell activation plays a central role in certaindebilitating autoimmune diseases such as rheumatoid arthritis, multiplesclerosis, and asthma. Thus presentation and recognition of antigenicepitopes presented by MHC molecules play a central role in mediatingimmune responses in multiple pathological conditions.

[0004] Tumor specific T cells, derived from cancer patients, will bindand lyse tumor cells. This specificity is based on their ability torecognize short amino acid sequences (epitopes) presented on the surfaceof the tumor cells by MHC class I and, in some cell types, class IImolecules. These epitopes are derived from the proteolytic degradationof intracellular proteins called tumor antigens encoded by genes thatare either uniquely or aberrantly expressed in tumor or cancer cells.

[0005] The availability of specific anti-tumor T cells has enabled theidentification of tumor antigens and subsequently the generation ofcancer vaccines designed to provoke an anti-tumor immune response.Anti-tumor T cells are localized within cancer patients, including inthe blood (where they can be found in the peripheral blood mononuclearcell fraction), in primary and secondary lymphoid tissue, e.g., thespleen, in ascites fluid in ovarian cancer patients (tumor associatedlymphocytes or TALs) or within the tumor itself (tumor infiltratinglymphocytes or TILs). Of these, TILs have been the most useful in theidentification of tumor antigens and tumor antigen-derived peptidesrecognized by T cells.

[0006] Conventional methods to generate TILs involve mincing tumorbiopsy tissue and culturing the cell suspension in vitro in the presenceof the T cell growth factor interleukin-2 (IL-2). Over a period ofseveral days, the combination of the tumor cells and IL-2 can stimulatethe proliferation of tumor specific T cells at the expense of tumorcells. In this way, the T cell population is expanded. The T cellsderived from the first expansion are subsequently mixed with eithermitomycin C-treated or irradiated tumor cells and cultured in vitro withIL2 to promote further proliferation and enrichment of tumor reactive Tcells. After several rounds of in vitro expansion, a potent anti-tumor Tcell population can be recovered and used to identify tumor antigens viaconventional but tedious expression cloning methodology. Kawakani Y. etal. (1994) Proc. Natl. Acad. Sci. USA 91(9):3515-3519.

[0007] This currently employed methodology used to generate tumorspecific T cells in vitro is unreliable and the antigens identified bythis method do not necessarily induce an anti-tumor immune response.Numerous experiments demonstrate that the encounter of antigens bymature T cells often results in the induction of tolerance because ofignorance, anergy or physical deletion. Pardoll (1998) Nature Med.4(5):525-531.

[0008] The ability of a particular peptide to function as a T cellepitope requires that it bind effectively to the antigen presentingdomain of an MHC molecule and also that it display an appropriate set ofamino acids that can be specifically recognized by a T cell receptormolecule. While it is possible to identify natural T cell epitopesderived from antigenic polypeptides, these peptide epitopes do notnecessarily represent antigens that are optimized for inducing aparticular immune response. In fact, it has been shown that it ispossible to improve the effectiveness of natural epitopes by introducingsingle amino or multiple acids substitutions that alter their sequence(Valmori et al. (2000) J. Immunol 164(2):1125-1131). Thus, delivery ofcarefully optimized synthetic peptide epitopes has the potential toprovide an improved method to induce a useful immune response.

[0009] The introduction into an animal of an antigen has been widelyused for the purposes of modulating the immune response, or lackthereof, to the antigen for a variety of purposes. These includevaccination against pathogens, induction of an immune response to acancerous cell, reduction of an allergic response, reduction of animmune response to a self antigen that occurs as a result of anautoimmune disorder, reduction of allograft rejection, and induction ofan immune response to a self antigen for the purpose of contraception.

[0010] In the treatment of cancer, a variety of immunotherapeuticapproaches have been taken to generate populations of cytotoxic Tlymphocytes which specifically recognize and lyse tumor cells. Many ofthese approaches depend in part on identifying and characterizingtumor-specific antigens.

[0011] More recently, certain pathogen- and tumor-related proteins havebeen immunologically mimicked with synthetic peptides whose amino acidsequence corresponds to that of an antigenic determinant domain of thepathogen- or tumor-related protein. Despite these advances, peptideimmunogens based on native sequences generally perform less thanoptimally with respect to inducing an immune response. Thus, a needexists for modified synthetic antigenic peptide epitopes with enhancedimmunomodulatory properties. This invention satisfies this need andprovides related advantages as well.

DISCLOSURE OF THE INVENTION

[0012] The present invention provides novel synthetic therapeuticcompounds. These compounds are designed to enhance binding to MHCmolecules and to enhance immunoregulatory properties relative to theirnatural counterparts. The synthetic compounds of the invention areuseful to modulate an immune response to the synthetic and naturallyoccurring compounds.

[0013] Further provided are polynucleotides encoding the compounds ofthe invention, gene delivery vehicles comprising these polynucleotidesand host cells comprising these polynucleotides.

[0014] In addition, the invention provides methods for inducing animmune response in a subject by delivering the compounds andcompositions of the invention, and delivering these in the context of anMHC molecule.

[0015] The compounds of the invention are also useful to generateantibodies that specifically recognize and bind to these molecules.These antibodies are further useful for immunotherapy when administeredto a subject.

[0016] The invention also provides immune effector cells raised in vivoor in vitro in the presence and at the expense of an antigen presentingcell that presents the peptide compositions of the invention in thecontext of an MHC molecule and a method of adoptive immunotherapycomprising administering an effective amount of these immune effectorcells to a subject.

[0017] Further provided by this invention is a composition comprising atleast two immunogenic ligands, wherein said immunogenic ligands areindividually characterized by an ability to elicit an immune responseagainst the same native ligand, and wherein said immunogenic ligand isselected from the group consisting of FLYKWHGFV (SEQ ID NO:3), FLHKVHFYV(SEQ ID NO:5), FLHKWHWVV (SEQ ID NO:7), FLHKWHWYV (SEQ ID NO:9),FLHKVHYLV (SEQ ID NO:11) and KHFKPHGFS (SEQ ID NO:13). The ligands canbe present in a carrier such as a pharmaceutically acceptable carrier.

[0018] Also provided by this invention is a host cell comprising atleast two immunogenic ligands, wherein said immunogenic ligands areindividually characterized by an ability to elicit an immune responseagainst the same native ligand, and wherein said immunogenic ligand isselected from the group consisting of FLYKWHGFV (SEQ ID NO:3), FLHKVHFYV(SEQ ID NO:5), FLHKWHWVV (SEQ ID NO:7),

[0019] FLHKWHWYV (SEQ ID NO:9), FLHKVHYLV (SEQ ID NO:11) and KHFKPHGFS(SEQ ID NO:13). In one aspect, the host cell is an antigen presentingcell and the immunogenic ligands are presented on the surface of thecell. In a further aspect, the antigen presenting cell is a dendriticcell. The host cells can be present in a carrier, such as apharmaceutically acceptable carrier.

[0020] Still further provided by this invention is a method for inducingan immune response in a subject, by delivering to the subject acomposition comprising an effective amount of two or more immunogenicligands, wherein each of said immunogenic ligands is characterized by anability to elicit an immune response against the same native ligand, andwherein said immunogenic ligand is selected from the group consisting ofFLYKWHGFV (SEQ ID NO:3), FLHKVHFYV (SEQ ID NO:5), FLHKWHWVV (SEQ IDNO:7), FLHKWHWYV (SEQ ID NO:9), FLHKVHYLV

[0021] (SEQ ID NO:11) and KHFKPHGFS (SEQ ID NO:13).

DESCRIPTION OF THE SEQUENCE LISTINGS

[0022] SEQ ID NO:1. The complete nucleotide sequence of a cDNA encodingthe human cancer antigen ATF4/CREB-2. The coding region extends fromnucleotide 882 through nucleotide 1937. The nucleotide and amino acidsequence also are available in GenBank under Accession No. NM 001675.

[0023] SEQ ID NO:2. The amino acid sequence of the native human cancerantigen ATF4/CREB-2. The compounds of the invention are variations basedon native peptide 42-50.

[0024] SEQ ID NO:3. The amino acid sequence of compound 1.

[0025] SEQ ID NO:4. The polynucleotide sequence encoding compound 1.

[0026] SEQ ID NO:5. The amino acid sequence of compound 2.

[0027] SEQ ID NO:6. The polynucleotide sequence encoding compound 2.

[0028] SEQ ID NO:7. The amino acid sequence of compound 3.

[0029] SEQ ID NO:8. The polynucleotide sequence encoding compound 3.

[0030] SEQ ID NO:9. The amino acid sequence of compound 4.

[0031] SEQ ID NO:10. The polynucleotide sequence encoding compound 4.

[0032] SEQ ID NO:11. The amino acid sequence of compound 5.

[0033] SEQ ID NO:12. The polynucleotide sequence encoding compound 5.

[0034] SEQ ID NO:13 The natural epitope of human cancer antigenATF4/CREB-2.

[0035] SEQ ID NO:14: The polynucleotide sequence encoding the humancancer antigen ATF4/CREB-2.

MODES OF CARRYING OUT THE INVENTION

[0036] Throughout this disclosure, various publications, patents andpublished patent specifications are referenced by an identifyingcitation. The disclosures of these publications, patents and publishedpatent specifications are hereby incorporated by reference into thepresent disclosure to more fully describe the state of the art to whichthis invention pertains.

[0037] The practice of the present invention employs, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature. These methods are described in thefollowing publications. See, e.g., Sambrook et al. MOLECULAR CLONING: ALABORATORY MANUAL, 2^(nd) edition (1989); CURRENT PROTOCOLS IN MOLECULARBIOLOGY (F. M. Ausubel et al. eds. (1987)); the series METHODS INENZYMOLOGY (Academic Press, Inc.); PCR: A PRACTICAL APPROACH (M.MacPherson et al. IRL Press at Oxford University Press (1991)); PCR 2: APRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G. R. Taylor eds.(1995)); ANTIBODIES, A LABORATORY MANUAL (Harlow and Lane eds. (1988));and ANIMAL CELL CULTURE (R. I. Freshney ed. (1987)).

[0038] Definitions

[0039] As used herein, certain terms may have the following definedmeanings.

[0040] As used in the specification and claims, the singular form “a,”“an” and “the” include plural references unless the context clearlydictates otherwise. For example, the term “a cell” includes a pluralityof cells, including mixtures thereof.

[0041] As used herein, the term “comprising” is intended to mean thatthe compositions and methods include the recited elements, but notexcluding others. “Consisting essentially of” when used to definecompositions and methods, shall mean excluding other elements of anyessential significance to the combination. Thus, a compositionconsisting essentially of the elements as defined herein would notexclude trace contaminants from the isolation and purification methodand pharmaceutically acceptable carriers, such as phosphate bufferedsaline, preservatives, and the like. “Consisting of” shall meanexcluding more than trace elements of other ingredients and substantialmethod steps for administering the compositions of this invention.Embodiments defined by each of these transition terms are within thescope of this invention.

[0042] A “native” or “natural” antigen is a polypeptide, protein or afragment which contains an epitope, which has been isolated from anatural biological source, and which can specifically bind to an antigenreceptor, in particular a T cell antigen receptor (TCR), in a subject.

[0043] The term “antigen” is well understood in the art and includessubstances which are immunogenic, i.e., immunogens, as well assubstances which induce immunological unresponsiveness, or anergy, i.e.,anergens.

[0044] An “altered antigen” is one having a primary sequence that isdifferent from that of the corresponding wild-type antigen. Alteredantigens can be made by synthetic or recombinant methods and include,but are not limited to, antigenic peptides that are differentiallymodified during or after translation, e.g., by phosphorylation,glycosylation, cross-linking, acylation, proteolytic cleavage, linkageto an antibody molecule, membrane molecule or other ligand. (Ferguson etal. (1988) Ann. Rev. Biochem. 57:285-320). A synthetic or alteredantigen of the invention is intended to bind to the same TCR as thenatural epitope.

[0045] A “self-antigen” also referred to herein as a native or wild-typeantigen is an antigenic peptide that induces little or no immuneresponse in the subject due to self-tolerance to the antigen. An exampleof a self-antigen is the melanoma specific antigen gp100.

[0046] The term “tumor associated antigen” or “TAA” refers to an antigenthat is associated with or specific to a tumor. Examples of known TAAsinclude gp100, MART and MAGE.

[0047] The terms “major histocompatibility complex” or “MHC” refers to acomplex of genes encoding cell-surface molecules that are required forantigen presentation to T cells and for rapid graft rejection. Inhumans, the MHC is also known as the “human leukocyte antigen” or “HLA”complex. The proteins encoded by the MHC are known as “MHC molecules”and are classified into class I and class II MHC molecules. Class I MHCincludes membrane heterodimeric proteins made up of an α chain encodedin the MHC noncovalently linked with the β2-microglobulin. Class I MHCmolecules are expressed by nearly all nucleated cells and have beenshown to function in antigen presentation to CD8+T cells. Class Imolecules include HLA-A, B, and C in humans. Class II MHC molecules alsoinclude membrane heterodimeric proteins consisting of noncovalentlyassociated α and β chains. Class II MHC molecules are known to functionin CD4+T cells and, in humans, include HLA-DP, -DQ, and DR. In apreferred embodiment, invention compositions and ligands can complexwith MHC molecules of any HLA type. Those of skill in the art arefamiliar with the serotypes and genotypes of the HLA. See:http://bimas.dcrt.nih.gov/cgi-bin/molbio/hla_coefficient_viewing_page.Rammensee H. G., Bachmann J., and Stevanovic S. MHC Ligands and PeptideMotifs (1997) Chapman & Hall Publishers; Schreuder G. M. Th. et al. TheHLA dictionary (1999) Tissue Antigens 54:409-437.

[0048] The term “antigen-presenting matrix”, as used herein, intends amolecule or molecules which can present antigen in such a way that theantigen can be bound by a T-cell antigen receptor on the surface of a Tcell. An antigen-presenting matrix can be on the surface of anantigen-presenting cell (APC), on a vesicle preparation of an APC, orcan be in the form of a synthetic matrix on a solid support such as abead or a plate. An example of a synthetic antigen-presenting matrix ispurified MHC class I molecules complexed to P2-microglobulin, multimersof such purified MHC class I molecules, purified MHC Class II molecules,or functional portions thereof, attached to a solid support.

[0049] The term “antigen presenting cells (APC)” refers to a class ofcells capable of presenting one or more antigens in the form ofantigen-MHC complex recognizable by specific effector cells of theimmune system, and thereby inducing an effective cellular immuneresponse against the antigen or antigens being presented. While manytypes of cells may be capable of presenting antigens on their cellsurface for T-cell recognition, only professional APCs have the capacityto present antigens in an efficient amount and further to activateT-cells for cytotoxic T-lymphocyte (CTL) responses. APCs can be intactwhole cells such as macrophages, B-cells and dendritic cells; or othermolecules, naturally occurring or synthetic, such as purified MHC classI molecules complexed to P2-microglobulin.

[0050] The term “dendritic cells (DC)” refers to a diverse population ofmorphologically similar cell types found in a variety of lymphoid andnon-lymphoid tissues (Steinman (1991) Ann. Rev. Immunol. 9:271-296).Dendritic cells constitute the most potent and preferred APCs in theorganism. A subset, if not all, of dendritic cells are derived from bonemarrow progenitor cells, circulate in small numbers in the peripheralblood and appear either as immature Langerhans' cells or terminallydifferentiated mature cells. While the dendritic cells can bedifferentiated from monocytes, they possess distinct phenotypes. Forexample, a particular differentiating marker, CD14 antigen, is not foundin dendritic cells but is possessed by monocytes.

[0051] Also, mature dendritic cells are not phagocytic, whereas themonocytes are strongly phagocytosing cells. It has been shown that DCsprovide all the signals necessary for T cell activation andproliferation.

[0052] The term “antigen presenting cell recruitment factors” or “APCrecruitment factors” include both intact, whole cells as well as othermolecules that are capable of recruiting antigen presenting cells.Examples of suitable APC recruitment factors include molecules such asinterleukin 4 (IL4), granulocyte macrophage colony stimulating factor(GM-CSF), Sepragel and macrophage inflammatory protein 3 alpha (MIP3α).These are available from Immunex, Schering-Plough and R&D Systems(Minneapolis, Minn.). They also can be recombinantly produced using themethods disclosed in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M.Ausubel et al., eds. (1987)). Peptides, proteins and compounds havingthe same biological activity as the above-noted factors are includedwithin the scope of this invention.

[0053] The term “immune effector cells” refers to cells capable ofbinding an antigen and which mediate an immune response. These cellsinclude, but are not limited to, T cells, B cells, monocytes,macrophages, NK cells and cytotoxic T lymphocytes (CTLs), for exampleCTL lines, CTL clones, and CTLs from tumor, inflammatory, or otherinfiltrates. Certain diseased tissue expresses specific antigens andCTLs specific for these antigens have been identified. For example,approximately 80% of melanomas express the antigen known as GP-100.

[0054] The term “immune effector molecule” as used herein, refers tomolecules capable of antigen-specific binding, and includes antibodies,T cell antigen receptors, and MHC Class I and Class II molecules.

[0055] A “naïve” immune effector cell is an immune effector cell thathas never been exposed to an antigen capable of activating that cell.Activation of naive immune effector cells requires both recognition ofthe peptide:MHC complex and the simultaneous delivery of a costimulatorysignal by a professional APC in order to proliferate and differentiateinto antigen-specific armed effector T cells.

[0056] “Immune response” broadly refers to the antigen-specificresponses of lymphocytes to foreign substances. Any substance that canelicit an immune response is said to be “immunogenic” and is referred toas an “immunogen”. All immunogens are antigens, however, not allantigens are immunogenic. An immune response of this invention can behumoral (via antibody activity) or cell-mediated (via T cellactivation).

[0057] The term “ligand” as used herein refers to any molecule thatbinds to a specific site on another molecule. In other words, the ligandconfers the specificity of the protein in a reaction with an immuneeffector cell. It is the ligand site within the protein that combinesdirectly with the complementary binding site on the immune effectorcell.

[0058] In a preferred embodiment, a ligand of the invention binds to anantigenic determinant or epitope on an immune effector cell, such as anantibody or a T cell receptor (TCR). A ligand may be an antigen,peptide, protein or epitope of the invention.

[0059] Invention ligands may bind to a receptor on an antibody. In oneembodiment, the ligand of the invention is about 4 to about 8 aminoacids in length.

[0060] Invention ligands may bind to a receptor on an MHC class Imolecule. In one embodiment, the ligand of the invention is about 7 toabout 11 amino acids in length.

[0061] Invention ligands may bind to a receptor on an MHC class IImolecule. In one embodiment, the ligand of the invention is about 10 toabout 20 amino acids long.

[0062] As used herein, the term “educated, antigen-specific immuneeffector cell”, is an immune effector cell as defined above, which haspreviously encountered an antigen. In contrast with its naivecounterpart, activation of an educated, antigen-specific immune effectorcell does not require a costimulatory signal. Recognition of thepeptide:MHC complex is sufficient.

[0063] “Activated”, when used in reference to a T cell, implies that thecell is no longer in G₀ phase, and begins to produce one or more ofcytotoxins, cytokines, and other related membrane-associated proteinscharacteristic of the cell type (e.g., CD8⁺ or CD4⁺), is capable ofrecognizing and binding any target cell that displays the particularantigen on its surface, and releasing its effector molecules.

[0064] In the context of the present invention, the term “recognized”intends that a composition of the invention, comprising one or moreligands, is recognized and bound by an immune effector cell wherein suchbinding initiates an effective immune response. Assays for determiningwhether a ligand is recognized by an immune effector cell are known inthe art and are described herein.

[0065] The term “preferentially recognized” intends that the specificityof a composition or ligand of the invention is restricted to immuneeffector cells that recognize and bind the native ligand.

[0066] The term “cross-reactive” is used to describe compounds of theinvention which are functionally overlapping. More particularly, theimmunogenic properties of a native ligand and/or immune effector cellsactivated thereby are shared to a certain extent by the altered ligandsuch that the altered ligand is “cross-reactive” with the native ligandand/or the immune effector cells activated thereby. For purposes of thisinvention, cross-reactivity is manifested at multiple levels: (i) at theligand level, e.g., the altered ligands can bind the TCR of and activatenative ligand CTLs; (ii) at the T cell level, i.e., altered ligands ofthe invention bind the TCR of and activate a population of T cells(distinct from the population of native ligand CTLs) which caneffectively target and lyse cells displaying the native ligand; and(iii) at the antibody level, e.g., “anti”-altered ligand antibodies candetect, recognize and bind the native ligand and initiate effectormechanisms in an immune response which ultimately result in eliminationof the native ligand from the host.

[0067] As used herein, the term “inducing an immune response in asubject” is a term well understood in the art and intends that anincrease of at least about 2-fold, more preferably at least about5-fold, more preferably at least about 10-fold, more preferably at leastabout 100-fold, even more preferably at least about 500-fold, even morepreferably at least about 1000-fold or more in an immune response to anantigen (or epitope) can be detected or measured, after introducing theantigen (or epitope) into the subject, relative to the immune response(if any) before introduction of the antigen (or epitope) into thesubject. An immune response to an antigen (or epitope), includes, but isnot limited to, production of an antigen-specific (or epitope-specific)antibody, and production of an immune cell expressing on its surface amolecule which specifically binds to an antigen (or epitope). Methods ofdetermining whether an immune response to a given antigen (or epitope)has been induced are well known in the art. For example,antigen-specific antibody can be detected using any of a variety ofimmunoassays known in the art, including, but not limited to, ELISA,wherein, for example, binding of an antibody in a sample to animmobilized antigen (or epitope) is detected with a detectably-labeledsecond antibody (e.g., enzyme-labeled mouse anti-human Ig antibody).

[0068] “Co-stimulatory molecules” are involved in the interactionbetween receptor-ligand pairs expressed on the surface of antigenpresenting cells and T cells. Research accumulated over the past severalyears has demonstrated convincingly that resting T cells require atleast two signals for induction of cytokine gene expression andproliferation (Schwartz R. H. (1990) Science 248:1349-1356 and JenkinsM. K. (1992) Immunol. Today 13:69-73). One signal, the one that confersspecificity, can be produced by interaction of the TCR/CD3 complex withan appropriate MHC/peptide complex. The second signal is not antigenspecific and is termed the “co-stimulatory” signal. This signal wasoriginally defined as an activity provided by bone-marrow-derivedaccessory cells such as macrophages and dendritic cells, the so called

[0069] “professional” APCs. Several molecules have been shown to enhanceco-stimulatory activity. These are heat stable antigen (HSA) (Liu Y. etal. (1992) J. Exp. Med. 175:437-445), chondroitin sulfate-modified MHCinvariant chain (Ii-CS) (Naujokas M. F. et al. (1993) Cell 74:257-268),intracellular adhesion molecule 1 (ICAM-1) (Van Seventer G. A. (1990) J.Immunol. 144:4579-4586), B7-1, and B7-2/B70 (Schwartz R. H. (1992) Cell71:1065-1068). These molecules each appear to assist co-stimulation byinteracting with their cognate ligands on the T cells. Co-stimulatorymolecules mediate co-stimulatory signal(s), which are necessary, undernormal physiological conditions, to achieve full activation of naive Tcells. One exemplary receptor-ligand pair is the B7 co-stimulatorymolecule on the surface of APCs and its counter-receptor CD28 or CTLA-4on T cells (Freeman et al. (1993) Science 262:909-911; Young et al.(1992) J. Clin. Invest. 90:229 and Nabavi et al. (1992) Nature360:266-268). Other important co-stimulatory molecules are CD40, CD54,CD80, and CD86. The term “co-stimulatory molecule” encompasses anysingle molecule or combination of molecules which, when acting togetherwith a peptide/MHC complex bound by a TCR on the surface of a T cell,provides a co-stimulatory effect which achieves activation of the T cellthat binds the peptide. The term thus encompasses B7, or otherco-stimulatory molecule(s) on an antigen-presenting matrix such as anAPC, fragments thereof (alone, complexed with another molecule(s), or aspart of a fusion protein) which, together with peptide/MHC complex,binds to a cognate ligand and results in activation of the T cell whenthe TCR on the surface of the T cell specifically binds the peptide.Co-stimulatory molecules are commercially available from a variety ofsources, including, for example, Beckman Coulter, Inc. (Fullerton,Calif.). It is intended, although not always explicitly stated, thatmolecules having similar biological activity as wild-type or purifiedco-stimulatory molecules (e.g., recombinantly produced or muteinsthereof) are intended to be used within the spirit and scope of theinvention.

[0070] As used herein, “solid phase support” or “solid support”, usedinterchangeably, is not limited to a specific type of support. Rather alarge number of supports are available and are known to one of ordinaryskill in the art. Solid phase supports include silica gels, resins,derivatized plastic films, glass beads, cotton, plastic beads, aluminagels. As used herein, “solid support” also includes syntheticantigen-presenting matrices, cells, and liposomes. A suitable solidphase support may be selected on the basis of desired end use andsuitability for various protocols. For example, for peptide synthesis,solid phase support may refer to resins such as polystyrene (e.g.,PAM-resin obtained from Bachem Inc., Peninsula Laboratories, etc.),POLYHIPE® resin (obtained from Aminotech, Canada), polyamide resin(obtained from Peninsula Laboratories), polystyrene resin grafted withpolyethylene glycol (TentaGel®, Rapp Polymere, Tubingen, Germany) orpolydimethylacrylamide resin (obtained from Milligen/Biosearch,California).

[0071] The term “immunomodulatory agent”, as used herein, is a molecule,a macromolecular complex, or a cell that modulates an immune responseand encompasses a synthetic antigenic peptide of the invention alone orin any of a variety of formulations described herein; a polypeptidecomprising a synthetic antigenic peptide of the invention; apolynucleotide encoding a peptide or polypeptide of the invention; asynthetic antigenic peptide of the invention bound to a Class I or aClass II MHC molecule on an antigen-presenting matrix, including an APCand a synthetic antigen-presenting matrix (in the presence or absence ofco-stimulatory molecule(s)); a synthetic antigenic peptide of theinvention covalently or non-covalently complexed to another molecule(s)or macromolecular structure; and an educated, antigen-specific immuneeffector cell which is specific for a peptide of the invention.

[0072] The term “modulate an immune response” includes inducing(increasing, eliciting) an immune response; and reducing (suppressing)an immune response. An immunomodulatory method (or protocol) is one thatmodulates an immune response in a subject.

[0073] As used herein, the term “cytokine” refers to any one of thenumerous factors that exert a variety of effects on cells, for example,inducing growth or proliferation. Non-limiting examples of cytokineswhich may be used alone or in combination in the practice of the presentinvention include, interleukin-2 (IL-2), stem cell factor (SCF),interleukin 3 (IL-3), interleukin 6 (IL-6), interleukin 12 (IL-12),G-CSF, granulocyte macrophage-colony stimulating factor (GM-CSF),interleukin-1 alpha (IL-1α), interleukin-11 (IL-11), MIP-11, leukemiainhibitory factor (LIF), c-kit ligand, thrombopoietin (TPO) and flt3ligand. The present invention also includes culture conditions in whichone or more cytokine is specifically excluded from the medium. Cytokinesare commercially available from several vendors such as, for example,Genzyme (Framingham, Mass.), Genentech (South San Francisco, Calif.),Amgen (Thousand Oaks, Calif.), R&D Systems (Minneapolis, Minn.) andImmunex (Seattle, Wash.). It is intended, although not always explicitlystated, that molecules having similar biological activity as wild-typeor purified cytokines (e.g., recombinantly produced or muteins thereof)are intended to be used within the spirit and scope of the invention.

[0074] The terms “polynucleotide” and “nucleic acid molecule” are usedinterchangeably to refer to polymeric forms of nucleotides of anylength. The polynucleotides may contain deoxyribonucleotides,ribonucleotides, and/or their analogs. Nucleotides may have anythree-dimensional structure, and may perform any function, known orunknown. The term “polynucleotide” includes, for example,single-stranded, double-stranded and triple helical molecules, a gene orgene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA,recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes, and primers. A nucleic acid molecule may alsocomprise modified nucleic acid molecules.

[0075] The term “peptide” is used in its broadest sense to refer to acompound of two or more subunit amino acids, amino acid analogs, orpeptidomimetics. The subunits may be linked by peptide bonds. In anotherembodiment, the subunit may be linked by other bonds, e.g. ester, ether,etc. As used herein the term “amino acid” refers to either naturaland/or unnatural or synthetic amino acids, including glycine and boththe D or L optical isomers, and amino acid analogs and peptidomimetics.A peptide of three or more amino acids is commonly called anoligopeptide if the peptide chain is short. If the peptide chain islong, the peptide is commonly called a polypeptide or a protein.

[0076] The term “genetically modified” means containing and/orexpressing a foreign gene or nucleic acid sequence which in turn,modifies the genotype or phenotype of the cell or its progeny. In otherwords, it refers to any addition, deletion or disruption to a cell'sendogenous nucleotides.

[0077] As used herein, “expression” refers to the process by whichpolynucleotides are transcribed into mRNA and translated into peptides,polypeptides, or proteins. If the polynucleotide is derived from genomicDNA, expression may include splicing of the mRNA, if an appropriateeukaryotic host is selected. Regulatory elements required for expressioninclude promoter sequences to bind RNA polymerase and transcriptioninitiation sequences for ribosome binding. For example, a bacterialexpression vector includes a promoter such as the lac promoter and fortranscription initiation the Shine-Dalgarno sequence and the start codonAUG (Sambrook et al. (1989) supra). Similarly, an eukaryotic expressionvector includes a heterologous or homologous promoter for RNA polymeraseII, a downstream polyadenylation signal, the start codon AUG, and atermination codon for detachment of the ribosome. Such vectors can beobtained commercially or assembled by the sequences described in methodswell known in the art, for example, the methods described below forconstructing vectors in general. “Under transcriptional control” is aterm well understood in the art and indicates that transcription of apolynucleotide sequence, usually a DNA sequence, depends on its beingoperatively linked to an element which contributes to the initiation of,or promotes, transcription. “Operatively linked” refers to ajuxtaposition wherein the elements are in an arrangement allowing themto function.

[0078] A “gene delivery vehicle” is defined as any molecule that cancarry inserted polynucleotides into a host cell. Examples of genedelivery vehicles are liposomes, biocompatible polymers, includingnatural polymers and synthetic polymers; lipoproteins; polypeptides;polysaccharides; lipopolysaccharides; artificial viral envelopes; metalparticles; and bacteria, or viruses, such as baculovirus, adenovirus andretrovirus, bacteriophage, cosmid, plasmid, fungal vectors and otherrecombination vehicles typically used in the art which have beendescribed for expression in a variety of eukaryotic and prokaryotichosts, and may be used for gene therapy as well as for simple proteinexpression.

[0079] “Gene delivery,” “gene transfer,” and the like as used herein,are terms referring to the introduction of an exogenous polynucleotide(sometimes referred to as a “transgene”) into a host cell, irrespectiveof the method used for the introduction. Such methods include a varietyof well-known techniques such as vector-mediated gene transfer (by,e.g., viral infection/transfection, or various other protein-based orlipid-based gene delivery complexes) as well as techniques facilitatingthe delivery of “naked” polynucleotides (such as electroporation, “genegun” delivery and various other techniques used for the introduction ofpolynucleotides). The introduced polynucleotide may be stably ortransiently maintained in the host cell. Stable maintenance typicallyrequires that the introduced polynucleotide either contains an origin ofreplication compatible with the host cell or integrates into a repliconof the host cell such as an extrachromosomal replicon (e.g., a plasmid)or a nuclear or mitochondrial chromosome. A number of vectors are knownto be capable of mediating transfer of genes to mammalian cells, as isknown in the art and described herein.

[0080] A “viral vector” is defined as a recombinantly produced virus orviral particle that comprises a polynucleotide to be delivered into ahost cell, either in vivo, ex vivo or in vitro. Examples of viralvectors include retroviral vectors, adenovirus vectors, adeno-associatedvirus vectors, alphavirus vectors and the like. Alphavirus vectors, suchas Semliki Forest virus-based vectors and Sindbis virus-based vectors,have also been developed for use in gene therapy and immunotherapy. See,Schlesinger and Dubensky (1999) Curr Opin Biotechnol. 5:434-439 and Zakset al. (1999) Nat. Med. 7:823-827. In aspects where gene transfer ismediated by a retroviral vector, a vector construct refers to thepolynucleotide comprising the retroviral genome or part thereof, and atherapeutic gene. As used herein, “retroviral mediated gene transfer” or“retroviral transduction” carries the same meaning and refers to theprocess by which a gene or nucleic acid sequences are stably transferredinto the host cell by virtue of the virus entering the cell andintegrating its genome into the host cell genome. The virus can enterthe host cell via its normal mechanism of infection or be modified suchthat it binds to a different host cell surface receptor or ligand toenter the cell. As used herein, retroviral vector refers to a viralparticle capable of introducing exogenous nucleic acid into a cellthrough a viral or viral-like entry mechanism.

[0081] Retroviruses carry their genetic information in the form of RNA;however, once the virus infects a cell, the RNA is reverse-transcribedinto the DNA form which integrates into the genomic DNA of the infectedcell. The integrated DNA form is called a provirus.

[0082] In aspects where gene transfer is mediated by a DNA viral vector,such as an adenovirus (Ad) or adeno-associated virus (AAV), a vectorconstruct refers to the polynucleotide comprising the viral genome orpart thereof, and a transgene. Adenoviruses (Ads) are a relatively wellcharacterized, homogenous group of viruses, including over 50 serotypes.See, e.g., WO 95/27071. Ads are easy to grow and do not requireintegration into the host cell genome. Recombinant Ad-derived vectors,particularly those that reduce the potential for recombination andgeneration of wild-type virus, have also been constructed. See, WO95/00655 and WO 95/11984. Wild-type AAV has high infectivity andspecificity integrating into the host cell's genome. See, Hermonat andMuzyczka (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470 and Lebkowski etal. (1988) Mol. Cell. Biol. 8:3988-3996.

[0083] Vectors that contain both a promoter and a cloning site intowhich a polynucleotide can be operatively linked are well known in theart. Such vectors are capable of transcribing RNA in vitro or in vivo,and are commercially available from sources such as Stratagene (LaJolla, Calif.) and Promega Biotech (Madison, Wis.). In order to optimizeexpression and/or in vitro transcription, it may be necessary to remove,add or alter 5′ and/or 3′ untranslated portions of the clones toeliminate extra, potential inappropriate alternative translationinitiation codons or other sequences that may interfere with or reduceexpression, either at the level of transcription or translation.Alternatively, consensus ribosome binding sites can be insertedimmediately 5′ of the start codon to enhance expression.

[0084] Gene delivery vehicles also include several non-viral vectors,including DNA/liposome complexes, and targeted viral protein-DNAcomplexes. Liposomes that also comprise a targeting antibody or fragmentthereof can be used in the methods of this invention. To enhancedelivery to a cell, the nucleic acid or proteins of this invention canbe conjugated to antibodies or binding fragments thereof which bind cellsurface antigens, e.g., TCR, CD3 or CD4.

[0085] “Hybridization” refers to a reaction in which one or morepolynucleotides react to form a complex that is stabilized via hydrogenbonding between the bases of the nucleotide residues. The hydrogenbonding may occur by Watson-Crick base pairing, Hoogstein binding, or inany other sequence-specific manner. The complex may comprise two strandsforming a duplex structure, three or more strands forming amulti-stranded complex, a single self-hybridizing strand, or anycombination of these. A hybridization reaction may constitute a step ina more extensive process, such as the initiation of a PCR reaction, orthe enzymatic cleavage of a polynucleotide by a ribozyme.

[0086] Examples of stringent hybridization conditions include:incubation temperatures of about 25° C. to about 37° C.; hybridizationbuffer concentrations of about 6×SSC to about 10×SSC; formamideconcentrations of about 0% to about 25%; and wash solutions of about6×SSC. Examples of moderate hybridization conditions include: incubationtemperatures of about 40° C. to about 50° C.; buffer concentrations ofabout 9×SSC to about 2×SSC; formamide concentrations of about 30% toabout 50%; and wash solutions of about 5×SSC to about 2×SSC. Examples ofhigh stringency conditions include: incubation temperatures of about 55°C. to about 68° C.; buffer concentrations of about 1×SSC to about0.1×SSC; formamide concentrations of about 55% to about 75%; and washsolutions of about 1×SSC, 0.1×SSC, or deionized water. In general,hybridization incubation times are from 5 minutes to 24 hours, with 1,2, or more washing steps, and wash incubation times are about 1, 2, or15 minutes. SSC is 0.15 M NaCl and 15 mM citrate buffer. It isunderstood that equivalents of SSC using other buffer systems can beemployed.

[0087] A polynucleotide or polynucleotide region (or a polypeptide orpolypeptide region) has a certain percentage (for example, 80%, 85%,90%, or 95%) of “sequence identity” to another sequence means that, whenaligned, that percentage of bases (or amino acids) are the same incomparing the two sequences. This alignment and the percent homology orsequence identity can be determined using software programs known in theart, for example those described in CURRENT PROTOCOLS IN MOLECULARBIOLOGY (F. M. Ausubel et al., eds., 1987) Supplement 30, section7.7.18, Table 7.7.1. Preferably, default parameters are used foralignment. A preferred alignment program is BLAST, using defaultparameters. In particular, preferred programs are BLASTN and BLASTP,using the following default parameters: Genetic code=standard;filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSLM62;Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant,GenBank+EMBL+DDBJ+PDB+GenBank CDStranslations+SwissProtein+SPupdate+PIR. Details of these programs can befound at the following Internet address:http://www.ncbi.nlm.nih.gov/cgi-bin/BLAST.

[0088] “In vivo” gene delivery, gene transfer, gene therapy and the likeas used herein, are terms referring to the introduction of a vectorcomprising an exogenous polynucleotide directly into the body of anorganism, such as a human or non-human mammal, whereby the exogenouspolynucleotide is introduced to a cell of such organism in vivo.

[0089] The term “isolated” means separated from constituents, cellularand otherwise, in which the polynucleotide, peptide, polypeptide,protein, antibody, or fragments thereof, are normally associated with innature. For example, with respect to a polynucleotide, an isolatedpolynucleotide is one that is separated from the 5′ and 3′ sequenceswith which it is normally associated in the chromosome. As is apparentto those of skill in the art, a non-naturally occurring polynucleotide,peptide, polypeptide, protein, antibody, or fragments thereof, does notrequire “isolation” to distinguish it from its naturally occurringcounterpart. In addition, a “concentrated”, “separated” or “diluted”polynucleotide, peptide, polypeptide, protein, antibody, or fragmentsthereof, is distinguishable from its naturally occurring counterpart inthat the concentration or number of molecules per volume is greater than“concentrated” or less than “separated” than that of its naturallyoccurring counterpart. A polynucleotide, peptide, polypeptide, protein,antibody, or fragments thereof, which differs from the naturallyoccurring counterpart in its primary sequence or for example, by itsglycosylation pattern, need not be present in its isolated form since itis distinguishable from its naturally occurring counterpart by itsprimary sequence, or alternatively, by another characteristic such asglycosylation pattern. Although not explicitly stated for each of theinventions disclosed herein, it is to be understood that all of theabove embodiments for each of the compositions disclosed below and underthe appropriate conditions, are provided by this invention. Thus, anon-naturally occurring polynucleotide is provided as a separateembodiment from the isolated naturally occurring polynucleotide. Aprotein produced in a bacterial cell is provided as a separateembodiment from the naturally occurring protein isolated from aeucaryotic cell in which it is produced in nature.

[0090] “Host cell,” “target cell” or “recipient cell” are intended toinclude any individual cell or cell culture which can be or have beenrecipients for vectors or the incorporation of exogenous nucleic acidmolecules, polynucleotides and/or proteins. It also is intended toinclude progeny of a single cell, and the progeny may not necessarily becompletely identical (in morphology or in genomic or total DNAcomplement) to the original parent cell due to natural, accidental, ordeliberate mutation. The cells may be procaryotic or eucaryotic, andinclude but are not limited to bacterial cells, yeast cells, animalcells, and mammalian cells, e.g., murine, rat, simian or human.

[0091] A “subject” is a vertebrate, preferably a mammal, more preferablya human. Mammals include, but are not limited to, murines, simians,humans, farm animals, sport animals, and pets.

[0092] A “control” is an alternative subject or sample used in anexperiment for comparison purpose. A control can be “positive” or“negative”. For example, where the purpose of the experiment is todetermine a correlation of an altered expression level of a gene with aparticular type of cancer, it is generally preferable to use a positivecontrol (a subject or a sample from a subject, carrying such alterationand exhibiting syndromes characteristic of that disease), and a negativecontrol (a subject or a sample from a subject lacking the alteredexpression and clinical syndrome of that disease).

[0093] The terms “cancer,” “neoplasm,” and “tumor,” used interchangeablyand in either the singular or plural form, refer to cells that haveundergone a malignant transformation that makes them pathological to thehost organism. Primary cancer cells (that is, cells obtained from nearthe site of malignant transformation) can be readily distinguished fromnon-cancerous cells by well-established techniques, particularlyhistological examination. The definition of a cancer cell, as usedherein, includes not only a primary cancer cell, but also any cellderived from a cancer cell ancestor. This includes metastasized cancercells, and in vitro cultures and cell lines derived from cancer cells.When referring to a type of cancer that normally manifests as a solidtumor, a “clinically detectable” tumor is one that is detectable on thebasis of tumor mass; e.g., by such procedures as CAT scan, magneticresonance imaging (MRI), X-ray, ultrasound or palpation. Biochemical orimmunologic findings alone may be insufficient to meet this definition.

[0094] “Suppressing” tumor growth indicates a growth state that iscurtailed compared to growth without contact with educated,antigen-specific immune effector cells described herein. Tumor cellgrowth can be assessed by any means known in the art, including, but notlimited to, measuring tumor size, determining whether tumor cells areproliferating using a ³H-thymidine incorporation assay, or countingtumor cells. “Suppressing” tumor cell growth means any or all of thefollowing states: slowing, delaying, and “suppressing” tumor growthindicates a growth state that is curtailed when stopping tumor growth,as well as tumor shrinkage.

[0095] The term “culturing” refers to the in vitro propagation of cellsor organisms on or in media of various kinds. It is understood that thedescendants of a cell grown in culture may not be completely identical(morphologically, genetically, or phenotypically) to the parent cell. By“expanded” is meant any proliferation or division of cells.

[0096] A “composition” is intended to mean a combination of active agentand another compound or composition, inert (for example, a detectableagent or label) or active, such as an adjuvant.

[0097] A “pharmaceutical composition” is intended to include thecombination of an active agent with a carrier, inert or active, makingthe composition suitable for diagnostic or therapeutic use in vitro, invivo or ex vivo.

[0098] As used herein, the term “pharmaceutically acceptable carrier”encompasses any of the standard pharmaceutical carriers, such as aphosphate buffered saline solution, water, and emulsions, such as anoil/water or water/oil emulsion, and various types of wetting agents.The compositions also can include stabilizers and preservatives. Forexamples of carriers, stabilizers and adjuvants, see Martin REMINGTON'SPHARM. SCI., 15th Ed. (Mack Publ. Co., Easton (1975)).

[0099] An “effective amount” is an amount sufficient to effectbeneficial or desired results. An effective amount can be administeredin one or more administrations, applications or dosages. The presentinvention provides compounds having the following structures:

[0100] The present invention also provides compositions that exhibitenhancing binding to MHC molecules and are cross-reactive with anduseful for modulating immune responses to the cognate native ligands andtheir corresponding native proteins.

[0101] This invention further provides compositions which are useful ascomponents of anti-cancer vaccines and to expand immune effector cellsthat are specific for cancers characterized by expression of the humancancer antigen ATF4/CREB-2. An example of this type of cancer is ovariancancer.

[0102] In one embodiment, the altered ligands of the invention havecomparable affinity for MHC binding as the native ligand. It has beendemonstrated that peptide:MHC class I binding properties correlate withimmunogenicity (Sette A. et al. (1994) Immunol. 153:5586; van der BurgS. H. et al. (1996) J. Immunol. 156:3308). In a preferred embodiment,altered ligands of the invention bind to a TCR with a higher affinitythan of that the “natural” ligand. Comparative binding of the native andaltered ligands of the invention to an MHC class I molecule can bemeasured by methods that are known in the art and include, but are notlimited to, calculating the affinity based on an algorithm (see, forexample, Parker et al. (1992) J. Immunol. 149:3580-3587) andexperimentally determining binding affinity (see, for example, Tan etal. (1997) J. Immunol. Meth. 209(1):25-36). For example, the relativebinding of a peptide to a class I molecule can be measured on the basisof binding of a radiolabeled standard peptide to detergent-solubilizedMHC molecules, using various concentrations of test peptides (e.g.,ranging from 100 mM to 1M). MHC class I heavy chain and P2-microglobulinare coincubated with a fixed concentration (e.g., 5 nM) radiolabeledstandard (control) peptide and various concentrations of a test peptidefor a suitable period of time (e.g., 2 hours to 72 hours) at roomtemperature in the presence of a mixture of protease inhibitors. Acontrol tube contains standard peptide and MHC molecules, but no testpeptide. The percent MHC-bound radioactivity is determined by gelfiltration. The IC₅₀ (concentration of test peptide which results in 50%inhibition of binding of control peptide) is calculated for eachpeptide. Additional methods for determining binding affinity to a TCRare known in the art and include, but are not limited to, thosedescribed in al-Ramadi et al. (1992) J. Immunol. 155(2):662-673; andZuegel et al. (1998) J. Immunol. 161(4):1705-1709.

[0103] In another embodiment, the altered ligands of the inventionelicit comparable antigen-specific T cell activation relative to theirnative ligand counterpart. In a preferred embodiment, altered ligands ofthe invention elicit a stronger antigen-specific T cell activationrelative to their native ligand counterpart. Methods for determiningimmunogenicity of invention ligands are known in the art and are furtherdescribed herein.

[0104] In one embodiment, compositions of the invention comprise two ormore immunogenic ligands of the invention. In one aspect, suchcompositions may comprise two or more copies of a single ligand. Inanother aspect, such compositions may comprise two or more ligands,wherein each ligand of said two or more ligands is distinct from allother ligands in said composition. In one embodiment, the two or moreimmunogenic ligands are covalently linked.

[0105] The present invention also provides novel synthetic antigenicpeptides designed for enhancing binding to MHC molecules and useful formodulating immune responses to the synthetic peptide epitope and thecorresponding native peptides from which they are derived. The syntheticantigenic peptide epitope sequences of the present invention differ fromtheir natural counterparts in that they contain alterations in aminoacid sequence, relative to the native sequence, in the MHC Class Ibinding domain which is designed to confer tighter binding to the MHC.They further contain mutations in the putative T cell receptor-bindingdomain designed to increase affinity for the T cell antigen receptor.These differences from the native sequence are designed to conferadvantages in the methods of the present invention over the nativesequence, in that the synthetic antigenic peptide epitopes of theinvention will have enhanced immunomodulatory properties.

[0106] This invention provides novel, synthetic antigenic peptidesequences, which are useful as components of anti-cancer vaccines and toexpand immune effector cells that are specific for cancers characterizedby expression of the human cancer antigen ATF4/CREB-2. The peptides,FLYKWHGFV (SEQ ID NO:3), FLHKVHFYV (SEQ ID NO:5), FLHKWHWVV (SEQ IDNO:7), FLHKWHWYV (SEQ ID NO:9), and FLHKVHYLV (SEQ ID NO:11) differ fromthe natural epitope KHFKPHGFS (SEQ ID NO:13) in that they containmutations in the putative HLA-A2 binding domain (amino acids 1, 2 and 9)and T cell receptor binding (TCR) domain (amino acid residues 3-8)conferring tighter binding to the MHC and TCR respectively. Binding ofsynthetic antigenic peptide of the invention to an MHC Class I moleculecan be measured by methods that are known in the art and include, butare not limited to, calculating the affinity based on an algorithm (see,for example, Parker et al. (1992) J. Immunol. 149:3580-3587); andexperimentally determining binding affinity (see, for example, Tan etal. (1997) J. Immunol. Meth. 209(1):25-36). For example, the relativebinding of a peptide to a Class I molecule can be measured on the basisof binding of a radiolabeled standard peptide to detergent-solubilizedMHC molecules, using various concentrations of test peptides (e.g.,ranging from 100 mM to 1 nM). MHC Class I heavy chain andβ2-microglobulin are coincubated with a fixed concentration (e.g., 5 nM)radiolabeled standard (control) peptide and various concentrations of atest peptide for a suitable period of time (e.g., 2 hours to 72 hours)at room temperature in the presence of a mixture of protease inhibitors.A control tube contains standard peptide and MHC molecules, but no testpeptide. The percent MHC-bound radioactivity is determined by gelfiltration. The IC50 (concentration of test peptide which results in 50%inhibition of binding of control peptide) is calculated for eachpeptide.

[0107] Synthetic peptides of the invention are designed to bind to a TCRwith a higher affinity than of that the “natural” sequence. Methods fordetermining binding affinity to a TCR are known in the art and include,but are not limited to, those described in al-Ramadi et al. (1992) J.Immunol. 155(2):662-673; and Zuegel et al. (1998) J. Immunol.161(4):1705-1709.

[0108] Further encompassed by the term “synthetic antigenic peptide” aremultimers (concatemers) of a synthetic antigenic peptide of theinvention, optionally including intervening amino acid sequences as wellas polypeptides comprising the sequences FLYKWHGFV (SEQ ID NO:3),FLHKVHFYV (SEQ ID NO:5), FLHKWHWVV (SEQ ID NO:7), FLHKWHWYV (SEQ IDNO:9), FLHKVHYLV (SEQ ID NO:11) and KHFKPHGFS (SEQ ID NO:13). Theinvention also provides polypeptides comprising these sequences whereinthe polypeptides are preferentially recognized by human cancer antigenATF4/CREB-2 cytotoxic T lymphocytes.

[0109] Polypeptides comprising the peptide sequences of the inventioncan be prepared by altering the sequence of polynucleotides that encodethe native human cancer antigen ATF4/CREB-2 polypeptide sequence. Thisis accomplished by methods of recombinant DNA technology well know tothose skilled in the art. For example, site directed mutagenesis may beperformed on recombinant polynucleotides encoding the native huan cancerantigen ATF4/CREB-2 sequence to introduce changes in the polynucleotidesequence so that the altered polynucleotide encodes the peptides of theinvention.

[0110] The proteins and polypeptides of this invention can be obtainedby chemical synthesis using a commercially available automated peptidesynthesizer such as those manufactured by Perkin Elner/AppliedBiosystems, Inc., Model 430A or 431A, Foster City, Calif., USA. Thesynthesized protein or polypeptide can be precipitated and furtherpurified, for example by high performance liquid chromatography (HPLC).Accordingly, this invention also provides a process for chemicallysynthesizing the proteins of this invention by providing the sequence ofthe protein and reagents, such as amino acids and enzymes and linkingtogether the amino acids in the proper orientation and linear sequence.

[0111] Alternatively, the proteins and polypeptides can be obtained bywell-known recombinant methods as described herein using the host celland vector systems described below.

[0112] Peptide analogues

[0113] It is well know to those skilled in the art that modificationscan be made to the peptides of the invention to provide them withaltered properties. As used herein the term “amino acid” refers toeither natural and/or unnatural or synthetic amino acids, includingglycine and both the D or L optical isomers, and amino acid analogs andpeptidomimetics. A peptide of three or more amino acids is commonlycalled an oligopeptide if the peptide chain is short. If the peptidechain is long, the peptide is commonly called a polypeptide or aprotein.

[0114] Peptides of the invention can be modified to include unnaturalamino acids. Thus, the peptides may comprise D-amino acids, acombination of D- and L-amino acids, and various “designer” amino acids(e.g., β-methyl amino acids, C-α-methyl amino acids, and N-α-methylamino acids, etc.) to convey special properties to peptides.Additionally, by assigning specific amino acids at specific couplingsteps, peptides with α-helices β turns, β sheets, γ-turns, and cyclicpeptides can be generated. Generally, it is believed that α-helicalsecondary structure or random secondary structure is preferred.

[0115] In a further embodiment, subunits of peptides that confer usefulchemical and structural properties will be chosen. For example, peptidescomprising D-amino acids will be resistant to L-amino acid-specificproteases in vivo. Modified compounds with D-amino acids may besynthesized with the amino acids aligned in reverse order to produce thepeptides of the invention as retro-inverso peptides. In addition, thepresent invention envisions preparing peptides that have better definedstructural properties, and the use of peptidomimetics, andpeptidomimetic bonds, such as ester bonds, to prepare peptides withnovel properties. In another embodiment, a peptide may be generated thatincorporates a reduced peptide bond, i.e., R-CH₂NH-R₂, where R₁, and R₂are amino acid residues or sequences. A reduced peptide bond may beintroduced as a dipeptide subunit. Such a molecule would be resistant topeptide bond hydrolysis, e.g., protease activity. Such molecules wouldprovide ligands with unique function and activity, such as extendedhalf-lives in vivo due to resistance to metabolic breakdown, or proteaseactivity. Furthermore, it is well known that in certain systemsconstrained peptides show enhanced functional activity (Hruby (1982)Life Sciences 31:189-199 and Hruby et al. (1990) Biochem J.268:249-262); the present invention provides a method to produce aconstrained peptide that incorporates random sequences at all otherpositions. Non-classical amino acids that induce conformationalconstraints.

[0116] The following non classical amino acids may be incorporated inthe peptides of the invention in order to introduce particularconformational motifs: 1,2,3,4-tetrahydroisoquinoline-3-carboxylate(Kazrnierski et al. (1991) J. Am. Chem. Soc. 113:2275-2283);(2S,3S)-methyl-phenylalanine, (2S,3R)-methyl-phenylalanine,(2R,3S)-methyl-phenylalanine and (2R,3R)-methyl-phenylalanine(Kazmierski and Hruby (1991) Tetrahedron Lett. 32(41):5769-5772);2-aminotetrahydronaphthalene-2-carboxylic acid (Landis (1989) Ph.D.Thesis, University of Ariz.);hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Miyake et al.(1989) J. Takeda Res. Labs. 43:53-76) histidine isoquinoline carboxylicacid (Zechel et al. (1991) Int. J. Pep. Protein Res. 38(2):131-138); andHIC (histidine cyclic urea), (Dharanipragada et al. (1993) Int. J. Pep.Protein Res. 42(1):68-77) and ((1992) Acta. Cryst., Crystal Struc. Comm.48(IV):1239-1241).

[0117] The following amino acid analogs and peptidomimetics may beincorporated into a peptide to induce or favor specific secondarystructures: LL-Acp (LL-3-amino-2-propenidone-6-carboxylic acid), aβ-turn inducing dipeptide analog (Kemp et al. (1985) J. Org. Chem.50:5834-5838); β-sheet inducing analogs (Kemp et al. (1988) TetrahedronLett. 29:5081-5082); β-turn inducing analogs (Kemp et al. (1988)Tetrahedron Lett. 29:5057-5060); α-helix inducing analogs (Kemp et al.(1988) Tetrahedron Lett. 29:4935-4938); γ-turn inducing analogs (Kemp etal. (1989) J. Org. Chem. 54:109:115); analogs provided by the followingreferences: Nagai and Sato (1985) Tetrahedron Lett. 26:647-650; andDiMaio et al. (1989) J. Chem. Soc. Perkin Trans. p. 1687; a Gly-Ala turnanalog (Kahn et al. (1989) Tetrahedron Lett. 30:2317); amide bondisostere (Clones et al. (1988) Tetrahedron Lett. 29:38S3-38S6);tretrazol (Zabrocki et al. (1988) J. Am. Chem. Soc. 110:587S-5880); DTC(Samanen et al. (1990) Int. J. Protein Pep. Res. 35:501:509); andanalogs taught in Olson et al. (1990) J. Am. Chem. Sci. 112:323-333 andGarvey et al. (1990) J. Org. Chem. 56:436. Conformationally restrictedmimetics of beta turns and beta bulges, and peptides containing them,are described in U.S. Pat. No. 5,440,013, issued Aug. 8, 1995 to Kahn.

[0118] A synthetic antigenic peptide epitope of the invention can beused in a variety of formulations, which may vary depending on theintended use.

[0119] A synthetic antigenic peptide epitope of the invention can becovalently or non-covalently linked (complexed) to various othermolecules, the nature of which may vary depending on the particularpurpose. For example, a peptide of the invention can be covalently ornon-covalently complexed to a macromolecular carrier, including, but notlimited to, natural and synthetic polymers, proteins, polysaccharides,polypeptides (amino acids), polyvinyl alcohol, polyvinyl pyrrolidone,and lipids. A peptide can be conjugated to a fatty acid, forintroduction into a liposome. U.S. Pat. No. 5,837,249. A syntheticpeptide of the invention can be complexed covalently or non-covalentlywith a solid support, a variety of which are known in the art. Asynthetic antigenic peptide epitope of the invention can be associatedwith an antigen-presenting matrix with or without co-stimulatorymolecules, as described in more detail below.

[0120] Examples of protein carriers include, but are not limited to,superantigens, serum albumin, tetanus toxoid, ovalbumin, thyroglobulin,myoglobulin, and immunoglobulin.

[0121] Peptide-protein carrier polymers may be formed using conventionalcross-linking agents such as carbodimides. Examples of carbodimides are1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide (CMC),1-ethyl-3-(3-dimethyaminopropyl) carbodiimide (EDC) and1-ethyl-3-(4-azonia-44-dimethylpentyl) carbodiimide.

[0122] Examples of other suitable cross-linking agents are cyanogenbromide, glutaraldehyde and succinic anhydride. In general, any of anumber of homo-bifunctional agents including a homo-bifunctionalaldehyde, a homo-bifunctional epoxide, a homo-bifunctional imido-ester,a homo-bifunctional N-hydroxysuccinimide ester, a homo-bifunctionalmaleimide, a homo-bifunctional alkyl halide, a homo-bifunctional pyridyldisulfide, a homo-bifunctional aryl halide, a homo-bifunctionalhydrazide, a homo-bifunctional diazonium derivative and ahomo-bifunctional photoreactive compound may be used. Also included arehetero-bifunctional compounds, for example, compounds having anamine-reactive and a sulfhydryl-reactive group, compounds with anamine-reactive and a photoreactive group and compounds with acarbonyl-reactive and a sulfhydryl-reactive group.

[0123] Specific examples of such homo-bifunctional cross-linking agentsinclude the bifunctional N-hydroxysuccinimide estersdithiobis(succinimidylpropionate), disuccinimidyl suberate, anddisuccinimidyl tartarate; the bifunctional imido-esters dimethyladipimidate, dimethyl pimelimidate, and dimethyl suberimidate; thebifunctional sulfhydryl-reactive crosslinkers1,4-di-[3′-(2′-pyridyldithio) propion-amido]butane, bismaleimidohexane,and bis-N-maleimido-1,8-octane; the bifunctional aryl halides1,5-difluoro-2,4-dinitrobenzene and4,4′-difluoro-3,3′-dinitrophenylsulfone; bifunctional photoreactiveagents such as bis-[b-(4-azidosalicylamido)ethyl]disulfide; thebifunctional aldehydes formaldehyde, malondialdehyde, succinaldehyde,glutaraldehyde, and adipaldehyde; a bifunctional epoxide such as1,4-butaneodiol diglycidyl ether; the bifunctional hydrazides adipicacid dihydrazide, carbohydrazide, and succinic acid dihydrazide; thebifunctional diazoniums o-tolidine, diazotized and bis-diazotizedbenzidine; the bifunctional alkylhalidesN1N′-ethylene-bis(iodoacetamide), N1N′-hexamethylene-bis(iodoacetamide),N1N′-undecamethylene-bis(iodoacetamide), as well as benzylhalides andhalomustards, such as ala′-diiodo-p-xylene sulfonic acid andtri(2-chloroethyl)amine, respectively.

[0124] Examples of common hetero-bifunctional cross-linking agents thatmay be used to effect the conjugation of proteins to peptides include,but are not limited to, SMCC(succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate), MBS(m-maleimidobenzoyl-N-hydroxysuccinimide ester), SIAB(N-succinimidyl(4-iodoacteyl)aminobenzoate), SMPB(succinimidyl-4-(p-maleimidophenyl)butyrate), GMBS(N-(γ-maleimidobutyryloxy)succinimide ester), MPBH(4-(4-N-maleimidopohenyl) butyric acid hydrazide), M2C2H(4-(N-maleimidomethyl) cyclohexane-1-carboxyl-hydrazide), SMPT(succinimidyloxycarbonyl-a-methyl-a-(2-pyridyldithio)toluene), and SPDP(N-succinimidyl 3-(2-pyridyldithio)propionate).

[0125] Cross-linking may be accomplished by coupling a carbonyl group toan amine group or to a hydrazide group by reductive amination.

[0126] Peptides of the invention also may be formulated as non-covalentattachment of monomers through ionic, adsorptive, or biospecificinteractions. Complexes of peptides with highly positively or negativelycharged molecules may be done through salt bridge formation under lowionic strength environments, such as in deionized water. Large complexescan be created using charged polymers such as poly-(L-glutamic acid) orpoly-(L-lysine) which contain numerous negative and positive charges,respectively. Adsorption of peptides may be done to surfaces such asmicroparticle latex beads or to other hydrophobic polymers, formingnon-covalently associated peptide-superantigen complexes effectivelymimicking cross-linked or chemically polymerized protein. Finally,peptides may be non-covalently linked through the use of biospecificinteractions between other molecules. For instance, utilization of thestrong affinity of biotin for proteins such as avidin or streptavidin ortheir derivatives could be used to form peptide complexes. Thesebiotin-binding proteins contain four binding sites that can interactwith biotin in solution or be covalently attached to another molecule.Wilchek (1988) Anal. Biochem. 171:1-32. Peptides can be modified topossess biotin groups using common biotinylation reagents such as theN-hydroxysuccinimidyl ester of D-biotin (NHS-biotin) which reacts withavailable amine groups on the protein. Biotinylated peptides then can beincubated with avidin or streptavidin to create large complexes. Themolecular mass of such polymers can be regulated through careful controlof the molar ratio of biotinylated peptide to avidin or streptavidin.

[0127] Also provided by this application are the peptides andpolypeptides described herein conjugated to a detectable agent for usein the diagnostic methods. For example, detectably labeled peptides andpolypeptides can be bound to a column and used for the detection andpurification of antibodies. They also are useful as immunogens for theproduction of antibodies, as described below.

[0128] The peptides of this invention also can be combined with variousliquid phase carriers, such as sterile or aqueous solutions,pharmaceutically acceptable carriers, suspensions and emulsions.Examples of non-aqueous solvents include propyl ethylene glycol,polyethylene glycol and vegetable oils. When used to prepare antibodies,the carriers also can include an adjuvant that is useful tonon-specifically augment a specific immune response. A skilled artisancan easily determine whether an adjuvant is required and select one.However, for the purpose of illustration only, suitable adjuvantsinclude, but are not limited to, Freund's Complete and Incomplete,mineral salts and polynucleotides.

[0129] This invention further provides polynucleotides encodingpolypeptides comprising the sequences FLYKWHGFV (SEQ ID NO:3), FLHKVHFYV(SEQ ID NO:5), FLHKWHWVV (SEQ ID NO:7), FLHKWHWYV (SEQ ID NO:9),FLHKVHYLV (SEQ ID NO:11) and KHFKPHGFS (SEQ ID NO:13) and thecomplements of these polynucleotides. As used herein, the term“polynucleotide” encompasses DNA, RNA and nucleic acid mimetics. Inaddition to the sequences identified above or their complements, thisinvention also provides the anti-sense polynucleotide stand, e.g.antisense RNA to these sequences or their complements. One can obtain anantisense RNA using the sequences provided in Seq. ID Nos. 4, 6, 8, 10,12 and 14 and the methodology described in Van der Krol, et al. (1988)BioTechniques 6:958.

[0130] The polynucleotides of this invention can be replicated usingPCR. PCR technology is the subject matter of U.S. Pat. Nos. 4,683,195;4,800,159; 4,754,065; and 4,683,202 and described in PCR: THE POLYMERASECHAIN REACTION (Mullis et al. eds, Birkhauser Press, Boston (1994)) andreferences cited therein.

[0131] Alternatively, one of skill in the art can use the sequencesprovided herein and a commercial DNA synthesizer to replicate the DNA.Accordingly, this invention also provides a process for obtaining thepolynucleotides of this invention by providing the linear sequence ofthe polynucleotide, appropriate primer molecules, chemicals such asenzymes and instructions for their replication and chemicallyreplicating or linking the nucleotides in the proper orientation toobtain the polynucleotides. In a separate embodiment, thesepolynucleotides are further isolated. Still further, one of skill in theart can insert the polynucleotide into a suitable replication vector andinsert the vector into a suitable host cell (procaryotic or eucaryotic)for replication and amplification. The DNA so amplified can be isolatedfrom the cell by methods well known to those of skill in the art. Aprocess for obtaining polynucleotides by this method is further providedherein as well as the polynucleotides so obtained.

[0132] RNA can be obtained by first inserting a DNA polynucleotide intoa suitable host cell. The DNA can be inserted by any appropriate method,e.g., by the use of an appropriate gene delivery vehicle (e.g.,liposome, plasmid or vector) or by electroporation. When the cellreplicates and the DNA is transcribed into RNA; the RNA can then beisolated using methods well known to those of skill in the art, forexample, as set forth in Sambrook et al. (1989) supra. For instance,mRNA can be isolated using various lytic enzymes or chemical solutionsaccording to the procedures set forth in Sambrook, et al. (1989) supraor extracted by nucleic-acid-binding resins following the accompanyinginstructions provided by manufactures.

[0133] Polynucleotides having at least 4 contiguous nucleotides, andmore preferably at least 5 or 6 contiguous nucleotides and mostpreferably at least 10 contiguous nucleotides, and exhibiting sequencecomplementarity or homology to the sequences encoding the amino acidsshown in SEQ ID NOS. 3, 5, 7, 9, 11 and 13, (e.g., SEQ ID NOS. 4, 6, 8,10, 12, or 14) find utility as hybridization probes.

[0134] It is known in the art that a “perfectly matched” probe is notneeded for a specific hybridization. Minor changes in probe sequenceachieved by substitution, deletion or insertion of a small number ofbases do not affect the hybridization specificity. In general, as muchas 20% base-pair mismatch (when optimally aligned) can be tolerated.Preferably, a probe useful for detecting the aforementioned mRNA is atleast about 80% identical to the homologous region of comparable sizecontained in the previously identified sequences (identified above)which correspond to previously characterized genes or in Seq. ID Nos. 1,4, 6, 8, 10, 12 or 14. More preferably, the probe is 85% identical tothe corresponding gene sequence after alignment of the homologousregion; even more preferably, it exhibits 90% identity.

[0135] These probes can be used in radioassays (e.g. Southern andNorthern blot analysis) to detect or monitor various cells or tissuecontaining these cells. The probes also can be attached to a solidsupport or an array such as a chip for use in high throughput screeningassays for the detection of expression of the gene corresponding to oneor more polynucleotide(s) of this invention. Accordingly, this inventionalso provides at least one probe identified as SEQ ID NOS. 4, 6, 8, 10,12 or 14, or the complement of one of these sequences, attached to asolid support for use in high throughput screens.

[0136] The polynucleotides of the present invention also can serve asprimers for the detection of genes or gene transcripts that areexpressed in APC, for example, to confirm transduction of thepolynucleotides into host cells. In this context, amplification meansany method employing a primer-dependent polymerase capable ofreplicating a target sequence with reasonable fidelity. Amplificationmay be carried out by natural or recombinant DNA-polymerases such as T7DNA polymerase, Klenow fragment of E. coli DNA polymerase, and reversetranscriptase. A preferred length of the primer is the same as thatidentified for probes, above.

[0137] The invention further provides the isolated polynucleotideoperatively linked to a promoter of RNA transcription, as well as otherregulatory sequences for replication and/or transient or stableexpression of the DNA or RNA. As used herein, the term “operativelylinked” means positioned in such a manner that the promoter will directtranscription of RNA off the DNA molecule. Examples of such promotersare SP6, T4 and T7. In certain embodiments, cell-specific promoters areused for cell-specific expression of the inserted polynucleotide.Vectors which contain a promoter or a promoter/enhancer, withtermination codons and selectable marker sequences, as well as a cloningsite into which an inserted piece of DNA can be operatively linked tothat promoter are well known in the art and commercially available. Forgeneral methodology and cloning strategies, see GENE EXPRESSIONTECHNOLOGY (Goeddel ed., Academic Press, Inc. (1991)) and referencescited therein and VECTORS: ESSENTIAL DATA SERIES (Gacesa and Ramji,eds., John Wiley & Sons, N.Y. (1994)), which contains maps, functionalproperties, commercial suppliers and a reference to GenEMBL accessionnumbers for various suitable vectors. Preferable, these vectors arecapable of transcribing RNA in vitro or in vivo.

[0138] Expression vectors containing these nucleic acids are useful toobtain host vector systems to produce proteins and polypeptides. It isimplied that these expression vectors must be replicable in the hostorganisms either as episomes or as an integral part of the chromosomalDNA. Suitable expression vectors include plasmids, viral vectors,including adenoviruses, adeno-associated viruses, retroviruses, cosmids,etc. Adenoviral vectors are particularly useful for introducing genesinto tissues in vivo because of their high levels of expression andefficient transformation of cells both in vitro and in vivo. When anucleic acid is inserted into a suitable host cell, e.g., a procaryoticor a eucaryotic cell and the host cell replicates, the protein can berecombinantly produced. Suitable host cells will depend on the vectorand can include mammalian cells, animal cells, human cells, simiancells, insect cells, yeast cells, and bacterial cells constructed usingwell known methods. See Sambrook, et al. (1989) supra. In addition tothe use of viral vector for insertion of exogenous nucleic acid intocells, the nucleic acid can be inserted into the host cell by methodswell known in the art such as transformation for bacterial cells;transfection using calcium phosphate precipitation for mammalian cells;DEAE-dextran; electroporation; or microinjection. See Sambrook et al.(1989) supra for this methodology. Thus, this invention also provides ahost cell, e.g. a mammalian cell, an animal cell (rat or mouse), a humancell, or a procaryotic cell such as a bacterial cell, containing apolynucleotide encoding a protein or polypeptide or antibody.

[0139] The present invention also provides delivery vehicles suitablefor delivery of a polynucleotide of the invention into cells (whether invivo, ex vivo, or in vitro). A polynucleotide of the invention can becontained within a cloning or expression vector. These vectors(especially expression vectors) can in turn be manipulated to assume anyof a number of forms which may, for example, facilitate delivery toand/or entry into a cell.

[0140] When the vectors are used for gene therapy in vivo or ex vivo, apharmaceutically acceptable vector is preferred, such as areplication-incompetent retroviral or adenoviral vector.Pharmaceutically acceptable vectors containing the nucleic acids of thisinvention can be further modified for transient or stable expression ofthe inserted polynucleotide. As used herein, the term “pharmaceuticallyacceptable vector” includes, but is not limited to, a vector or deliveryvehicle having the ability to selectively target and introduce thenucleic acid into dividing cells. An example of such a vector is a“replication-incompetent” vector defined by its inability to produceviral proteins, precluding spread of the vector in the infected hostcell. An example of a replication-incompetent retroviral vector is LNL6(Miller A. D. et al. (1989) BioTechniques 7:980-990). The methodology ofusing replication-incompetent retroviruses for retroviral-mediated genetransfer of gene markers is well established (Correll et al. (1989)Proc. Natl. Acad. Sci. USA 86:8912; Bordignon (1989) Proc. Natl. Acad.Sci. USA 86:8912-52; Culver K. (1991) Proc. Natl. Acad. Sci. USA88:3155; and Rill D. R. (1991) Blood 79(10):2694-2700).

[0141] These isolated host cells containing the polynucleotides of thisinvention are useful for the recombinant replication of thepolynucleotides and for the recombinant production of peptides.Alternatively, the cells may be used to induce an immune response in asubject in the methods described herein. When the host cells are antigenpresenting cells, they can be used to expand a population of immuneeffector cells such as tumor infiltrating lymphocytes which in turn areuseful in adoptive immunotherapies.

[0142] Also provided by this invention is an antibody capable ofspecifically forming a complex with the polypeptides of this invention.The term “antibody” includes polyclonal antibodies and monoclonalantibodies. The antibodies include, but are not limited to mouse, rat,and rabbit or human antibodies. The antibodies are useful to identifyand purify polypeptides and APCs expressing the polypeptides.

[0143] Laboratory methods for producing polyclonal antibodies andmonoclonal antibodies, as well as deducing their corresponding nucleicacid sequences, are known in the art, see Harlow and Lane (1988) supraand Sambrook et al. (1989) supra. The monoclonal antibodies of thisinvention can be biologically produced by introducing protein or afragment thereof into an animal, e.g., a mouse or a rabbit. The antibodyproducing cells in the animal are isolated and fused with myeloma cellsor hetero-myeloma cells to produce hybrid cells or hybridomas.Accordingly, the hybridoma cells producing the monoclonal antibodies ofthis invention also are provided.

[0144] Thus, using the protein or fragment thereof, and well knownmethods, one of skill in the art can produce and screen the hybridomacells and antibodies of this invention for antibodies having the abilityto bind the proteins or polypeptides.

[0145] If a monoclonal antibody being tested binds with the protein orpolypeptide, then the antibody being tested and the antibodies providedby the hybridomas of this invention are equivalent. It also is possibleto determine without undue experimentation, whether an antibody has thesame specificity as the monoclonal antibody of this invention bydetermining whether the antibody being tested prevents a monoclonalantibody of this invention from binding the protein or polypeptide withwhich the monoclonal antibody is normally reactive. If the antibodybeing tested competes with the monoclonal antibody of the invention asshown by a decrease in binding by the monoclonal antibody of thisinvention, then it is likely that the two antibodies bind to the same ora closely related epitope. Alternatively, one can pre-incubate themonoclonal antibody of this invention with a protein with which it isnormally reactive, and determine if the monoclonal antibody being testedis inhibited in its ability to bind the antigen. If the monoclonalantibody being tested is inhibited then, in all likelihood, it has thesame, or a closely related, epitopic specificity as the monoclonalantibody of this invention.

[0146] The term “antibody” also is intended to include antibodies of allisotypes. Particular isotypes of a monoclonal antibody can be preparedeither directly by selecting from the initial fusion, or preparedsecondarily, from a parental hybridoma secreting a monoclonal antibodyof different isotype by using the sib selection technique to isolateclass switch variants using the procedure described in Steplewski et al.(1985) Proc. Natl. Acad. Sci. USA 82:8653 or Spira et al. (1984) J.Immunol. Meth. 74:307.

[0147] This invention also provides biological active fragments of thepolyclonal and monoclonal antibodies described above. These “antibodyfragments” retain some ability to selectively bind with its antigen orimmunogen. Such antibody fragments can include, but are not limited to:

[0148] (1) Fab,

[0149] (2) Fab′,

[0150] (3) F(ab′)₂,

[0151] (4) Fv, and

[0152] (5) SCA

[0153] A specific example of “a biologically active antibody fragment”is a CDR region of the antibody. Methods of making these fragments areknown in the art, see for example, Harlow and Lane (1988) supra.

[0154] The antibodies of this invention also can be modified to createchimeric antibodies and humanized antibodies (Oi et al. (1986)BioTechniques 4(3):214). Chimeric antibodies are those in which thevarious domains of the antibodies' heavy and light chains are coded forby DNA from more than one species.

[0155] The isolation of other hybridomas secreting monoclonal antibodieswith the specificity of the monoclonal antibodies of the invention canalso be accomplished by one of ordinary skill in the art by producinganti-idiotypic antibodies (Herlyn et al. (1986) Science 232:100). Ananti-idiotypic antibody is an antibody which recognizes uniquedeterminants present on the monoclonal antibody produced by thehybridoma of interest.

[0156] Idiotypic identity between monoclonal antibodies of twohybridomas demonstrates that the two monoclonal antibodies are the samewith respect to their recognition of the same epitopic determinant.Thus, by using antibodies to the epitopic determinants on a monoclonalantibody it is possible to identify other hybridomas expressingmonoclonal antibodies of the same epitopic specificity.

[0157] It is also possible to use the anti-idiotype technology toproduce monoclonal antibodies which mimic an epitope. For example, ananti-idiotypic monoclonal antibody made to a first monoclonal antibodywill have a binding domain in the hypervariable region which is themirror image of the epitope bound by the first monoclonal antibody.Thus, in this instance, the anti-idiotypic monoclonal antibody could beused for immunization for production of these antibodies.

[0158] As used in this invention, the term “epitope” is meant to includeany determinant having specific affinity for the monoclonal antibodiesof the invention. Epitopic determinants usually consist of chemicallyactive surface groupings of molecules such as amino acids or sugar sidechains and usually have specific three dimensional structuralcharacteristics, as well as specific charge characteristics.

[0159] The antibodies of this invention can be linked to a detectableagent or label. There are many different labels and methods of labelingknown to those of ordinary skill in the art.

[0160] The coupling of antibodies to low molecular weight haptens canincrease the sensitivity of the assay. The haptens can then bespecifically detected by means of a second reaction. For example, it iscommon to use haptens such as biotin, which reacts avidin, ordinitropherryl, pyridoxal, and fluorescein, which can react withspecific anti-hapten antibodies. See Harlow and Lane (1988) supra.

[0161] The monoclonal antibodies of the invention also can be bound tomany different carriers. Thus, this invention also provides compositionscontaining the antibodies and another substance, active or inert.Examples of well-known carriers include glass, polystyrene,polypropylene, polyethylene, dextran, nylon, amylases, natural andmodified celluloses, polyacrylamides, agaroses and magnetite. The natureof the carrier can be either soluble or insoluble for purposes of theinvention. Those skilled in the art will know of other suitable carriersfor binding monoclonal antibodies, or will be able to ascertain such,using routine experimentation.

[0162] Compositions containing the antibodies, fragments thereof or celllines which produce the antibodies, are encompassed by this invention.When these compositions are to be used pharmaceutically, they arecombined with a pharmaceutically acceptable carrier.

[0163] In another embodiment the present invention provides a method ofinducing an immune response comprising delivering the compounds andcompositions of the invention in the context of an MHC molecule. Thus,the polypeptides of this invention can be pulsed into antigen presentingcells using the methods described herein. Antigen-presenting cells,include, but are not limited to dendritic cells (DCs),monocytes/macrophages, B lymphocytes or other cell type(s) expressingthe necessary MHC/co-stimulatory molecules. The methods described belowfocus primarily on DCs which are the most potent, preferred APCs. Thesehost cells containing the polypeptides or proteins are further provided.

[0164] Isolated host cells which present the polypeptides of thisinvention in the context of MHC molecules are further useful to expandand isolate a population of educated, antigen-specific immune effectorcells. The immune effector cells, e.g., cytotoxic T lymphocytes, areproduced by culturing naïve immune effector cells withantigen-presenting cells which present the polypeptides in the contextof MHC molecules on the surface of the APCs. The population can bepurified using methods known in the art, e.g., FACS analysis or ficol1gradient. The methods to generate and culture the immune effector cellsas well as the populations produced thereby also are the inventor'scontribution and invention. Pharmaceutical compositions comprising thecells and pharmaceutically acceptable carriers are useful in adoptiveimmunotherapy. Prior to administration in vivo, the immune effectorcells are screened in vitro for their ability to lyse tumor cellsexpressing the human cancer antigen ATF4/CREB-2, e.g., ovarian cancer.

[0165] In one embodiment, the immune effector cells and/or the APCs aregenetically modified. Using standard gene transfer, genes coding forco-stimulatory molecules and/or stimulatory cytokines can be insertedprior to, concurrent to or subsequent to expansion of the immuneeffector cells.

[0166] This invention also provides methods of inducing an immuneresponse in a subject, comprising administering to the subject aneffective amount of the polypeptides described above under theconditions that induce an immune response to the polypeptide. Thepolypeptides can be administered in formulations or as polynucleotidesencoding the polypeptides. The polynucleotides can be administered in agene delivery vehicle or by inserting into a host cell which in turnrecombinantly transcribes, translates and processed the encodedpolypeptide. Isolated host cells containing the polynucleotides of thisinvention in a pharmaceutically acceptable carrier can thereforecombined with appropriate and effective amount of an adjuvant, cytokineor co-stimulatory molecule for an effective vaccine regimen. In oneembodiment, the host cell is an APC such as a dendritic cell. The hostcell can be further modified by inserting of a polynucleotide coding foran effective amount of either or both a cytokine and/or a co-stimulatorymolecule.

[0167] The methods of this invention can be further modified byco-administering an effective amount of a cytokine or co-stimulatorymolecule to the subject.

[0168] This invention also provides compositions containing any of theabove-mentioned proteins, polypeptides, polynucleotides, vectors, cells,antibodies and fragments thereof, and an acceptable solid or liquidcarrier. When the compositions are used pharmaceutically, they arecombined with a “pharmaceutically acceptable carrier” for diagnostic andtherapeutic use. These compositions also can be used for the preparationof medicaments for the diagnosis and treatment of diseases such ascancer.

[0169] The following materials and methods are intended to illustrate,but not limit this invention and to illustrate how to make and use theinventions described above.

[0170] Materials and Methods

[0171] Production of the Polypeptides of the Invention

[0172] Most preferably, isolated peptides of the present invention canbe synthesized using an appropriate solid state synthetic procedure.Steward and Young, SOLID PHASE PEPTIDE SYNTHESIS, Freemantle, SanFrancisco, Calif. (1968). A preferred method is the Merrifield process.See, Merrifield (1967) Recent Progress in Hormone Res. 23:451. Theantigenic activity of these peptides may conveniently be tested using,for example, the assays as described herein.

[0173] Once an isolated peptide of the invention is obtained, it may bepurified by standard methods including chromatography (e.g., ionexchange, affinity, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for proteinpurification. For immuno-affinity chromatography, an epitope may beisolated by binding it to an affinity column comprising antibodies thatwere raised against that peptide, or a related peptide of the invention,and were affixed to a stationary support.

[0174] Alternatively, affinity tags such as hexa-His (Invitrogen),Maltose binding domain (New England Biolabs), influenza coat sequence(Kolodziej et al. (1991) Meth. Enzymol. 194:508-509), andglutathione-S-transferase can be attached to the peptides of theinvention to allow easy purification by passage over an appropriateaffinity column. Isolated peptides can also be physically characterizedusing such techniques as proteolysis, nuclear magnetic resonance, andx-ray crystallography.

[0175] Also included within the scope of the invention are antigenicpeptides that are differentially modified during or after translation,e.g., by phosphorylation, glycosylation, cross-linking, acylation,proteolytic cleavage, linkage to an antibody molecule, membrane moleculeor other ligand, (Ferguson et al. (1988) Ann. Rev. Biochem. 57:285-320).

[0176] Isolation, Culturing and Expansion of APCs, Including DendriticCells

[0177] The following is a brief description of two fundamentalapproaches for the isolation of APC. These approaches involve (1)isolating bone marrow precursor cells (CD34+) from blood and stimulatingthem to differentiate into APC; or (2) collecting the precommitted APCsfrom peripheral blood. In the first approach, the patient must betreated with cytokines such as GM-CSF to boost the number of circulatingCD34+stem cells in the peripheral blood.

[0178] The second approach for isolating APCs is to collect therelatively large numbers of precommitted APCs already circulating in theblood. Previous techniques for isolating committed APCs from humanperipheral blood have involved combinations of physical procedures suchas metrizamide gradients and adherence/non-adherence steps (FreudenthalP. S. et al. (1990) Proc. Natl. Acad. Sci. USA 87:7698-7702); Percollgradient separations (Mehta-Damani et al. (1994) J. Immunol.153:996-1003); and fluorescence activated cell sorting techniques(Thomas R. et al. (1993) J. Immunol. 151:6840-6852).

[0179] One technique for separating large numbers of cells from oneanother is known as countercurrent centrifugal elutriation (CCE). Inthis technique, cells are subject to simultaneous centrifugation and awashout stream of buffer that is constantly increasing in flow rate. Theconstantly increasing countercurrent flow of buffer leads to fractionalcell separations that are largely based on cell size.

[0180] In one aspect of the invention, the APC are precommitted ormature dendritic cells which can be isolated from the white blood cellfraction of a mammal, such as a murine, simian or a human (See, e.g., WO96/23060). The white blood cell fraction can be from the peripheralblood of the mammal. This method includes the following steps: (a)providing a white blood cell fraction obtained from a mammalian sourceby methods known in the art such as leukophoresis; (b) separating thewhite blood cell fraction of step (a) into four or more subfractions bycountercurrent centrifugal elutriation; (c) stimulating conversion ofmonocytes in one or more fractions from step (b) to dendritic cells bycontacting the cells with calcium ionophore, GM-CSF and IL-13 or GM-CSFand IL-4, (d) identifying the dendritic cell-enriched fraction from step(c); and (e) collecting the enriched fraction of step (d), preferably atabout 4° C. One way to identify the dendritic cell-enriched fraction isby fluorescence-activated cell sorting. The white blood cell fractioncan be treated with calcium ionophore in the presence of othercytokines, such as recombinant (rh) rhIL-12, rhGM-CSF, or rhIL-4. Thecells of the white blood cell fraction can be washed in buffer andsuspended in Ca⁺⁺/Mg₊₊ free media prior to the separating step. Thewhite blood cell fraction can be obtained by leukapheresis. Thedendritic cells can be identified by the presence of at least one of thefollowing markers: HLA-DR, HLA-DQ, or B7.2, and the simultaneous absenceof the following markers: CD3, CD14, CD16, 56, 57, and CD 19, 20.Monoclonal antibodies specific to these cell surface markers arecommercially available.

[0181] More specifically, the method requires collecting an enrichedcollection of white cells and platelets from leukapheresis that is thenfurther fractionated by countercurrent centrifugal elutriation (CCE)(Abrahamsen T. G. et al. (1991) J. Clin. Apheresis. 6:48-53). Cellsamples are placed in a special elutriation rotor. The rotor is thenspun at a constant speed of, for example, 3000 rpm. Once the rotor hasreached the desired speed, pressurized air is used to control the flowrate of cells. Cells in the elutriator are subjected to simultaneouscentrifugation and a washout stream of buffer that is constantlyincreasing in flow rate. This results in fractional cell separationsbased largely but not exclusively on differences in cell size.

[0182] Quality control of APC and more specifically DC collection andconfirmation of their successful activation in culture is dependent upona simultaneous multi-color FACS analysis technique which monitors bothmonocytes and the dendritic cell subpopulation as well as possiblecontaminant T lymphocytes. It is based upon the fact that DCs do notexpress the following markers: CD3 (T cell); CD14 (monocyte); CD16, 56,57 (NK/LAK cells); CD19, 20 (B cells). At the same time, DCs do expresslarge quantities of HLA-DR, significant HLA-DQ and B7.2 (but little orno B7.1) at the time they are circulating in the blood (in addition theyexpress Leu M7 and M9, myeloid markers which are also expressed bymonocytes and neutrophils).

[0183] When combined with a third color reagent for analysis of deadcells, propridium iodide (PI), it is possible to make positiveidentification of all cell subpopulations (see Table 1): TABLE 1 FACSanalysis of fresh peripheral cell subpopulations Color #1 Color #2 Color#3 Cocktail HLA-DR PI 3/14/16/19/20/56/57 Live Dendritic cells NegativePositive Negative Live Monocytes Positive Positive Negative LiveNeutrophils Negative Negative Negative Dead Cells Variable VariablePositive

[0184] Additional markers can be substituted for additional analysis:

[0185] Color #1: CD3 alone, CD14 alone, etc.; Leu M7 or Leu M9;anti-Class I, etc.

[0186] Color #2: HLA-DQ, B7.1, B7.2, CD25 (IL2r), ICAM, LFA-3, etc.

[0187] The goal of FACS analysis at the time of collection is to confirmthat the DCs are enriched in the expected fractions, to monitorneutrophil contamination, and to make sure that appropriate markers areexpressed. This rapid bulk collection of enriched DCs from humanperipheral blood, suitable for clinical applications, is absolutelydependent on the analytic FACS technique described above for qualitycontrol. If need be, mature DCs can be immediately separated frommonocytes at this point by fluorescent sorting for “cocktail negative”cells. It may not be necessary to routinely separate DCs from monocytesbecause, as will be detailed below, the monocytes themselves are stillcapable of differentiating into DCs or functional DC-like cells inculture.

[0188] Once collected, the DC rich/monocyte APC fractions (usually 150through 190) can be pooled and cryopreserved for future use, orimmediately placed in short term culture.

[0189] Alternatively, others have reported a method for upregulating(activating) dendritic cells and converting monocytes to an activateddendritic cell phenotype. This method involves the addition of calciumionophore to the culture media convert monocytes into activateddendritic cells. Adding the calcium ionophore A23187, for example, atthe beginning of a 24-48 hour culture period resulted in uniformactivation and dendritic cell phenotypic conversion of the pooled“monocyte plus DC” fractions: characteristically, the activatedpopulation becomes uniformly CD14 (Leu M3) negative, and upregulatesHLA-DR, HLA-DQ, ICAM-1, B7.1, and B7.2. Furthermore, this activated bulkpopulation functions as well on a small numbers basis as a furtherpurified.

[0190] Specific combination(s) of cytokines have been used successfullyto amplify (or partially substitute) for the activation/conversionachieved with calcium ionophore: these cytokines include but are notlimited to purified or recombinant (“rh”) rhGM-CSF, rhIL-2, and rhIL-4.Each cytokine when given alone is inadequate for optimal upregulation.

[0191] Presentation of Antigen to the APC

[0192] For purposes of immunization, the antigenic peptides (Nos. 3, 5,7, 9, 11 and 13) can be delivered to antigen-presenting cells asprotein/peptide or in the form of cDNA encoding the protein/peptide.Antigen-presenting cells (APCs) can consist of dendritic cells (DCs),monocytes/macrophages, B lymphocytes or other cell type(s) expressingthe necessary MHC/co-stimulatory molecules. The methods described belowfocus primarily on DCs which are the most potent, preferred APCs.

[0193] Pulsing is accomplished in vitro/ex vivo by exposing APCs to theantigenic protein or peptide(s) of this invention. The protein orpeptide(s) are added to APCs at a concentration of 1-10 μm forapproximately 3 hours. Pulsed APCs can subsequently be administered tothe host via an intravenous, subcutaneous, intranasal, intramuscular orintraperitoneal route of delivery.

[0194] Protein/peptide antigen can also be delivered in vivo withadjuvant via the intravenous, subcutaneous, intranasal, intramuscular orintraperitoneal route of delivery.

[0195] Paglia et al. (1996) J. Exp. Med. 183:317-322 has shown that APCincubated with whole protein in vitro were recognized by MHC classI-restricted CTLs, and that immunization of animals with these APCs ledto the development of antigen-specific CTLs in vivo. In addition,several different techniques have been described which lead to theexpression of antigen in the cytosol of APCs, such as DCs. These include(1) the introduction into the APCs of RNA isolated from tumor cells, (2)infection of APCs with recombinant vectors to induce endogenousexpression of antigen, and (3) introduction of tumor antigen into the DCcytosol using liposomes. (See Boczkowski D. et al. (1996) J. Exp. Med.184:465-472; Rouse et al. (1994) J. Virol. 68:5685-5689; and Nair et al.(1992) J. Exp. Med. 175:609-612).

[0196] Foster Antigen Presenting Cells

[0197] Foster antigen presenting cells are particularly useful as targetcells. Foster APCs are derived from the human cell line 174xCEM.T2,referred to as T2, which contains a mutation in its antigen processingpathway that restricts the association of endogenous peptides with cellsurface MHC class I molecules (Zweerink et al. (1993) J. Immunol.150:1763-1771). This is due to a large homozygous deletion in the MHCclass II region encompassing the genes TAP1, TAP2, LMP1, and LMP2, whichare required for antigen presentation to MHC class 1-restrictedCD8+CTLs. In effect, only “empty” MHC class I molecules are presented onthe surface of these cells. Exogenous peptide added to the culturemedium binds to these MHC molecules provided that the peptide containsthe allele-specific binding motif. These T2 cells are referred to hereinas “foster” APCs. They can be used in conjunction with this invention topresent antigen(s).

[0198] Transduction of T2 cells with specific recombinant MHC allelesallows for redirection of the MHC restriction profile. Librariestailored to the recombinant allele will be preferentially presented bythem because the anchor residues will prevent efficient binding to theendogenous allele.

[0199] High level expression of MHC molecules makes the APC more visibleto the CTLs. Expressing the MHC allele of interest in T2 cells using apowerful transcriptional promoter (e.g., the CMV promoter) results in amore reactive APC (most likely due to a higher concentration of reactiveMHC-peptide complexes on the cell surface).

[0200] Immunogenicity Assays.

[0201] The immunogenicity of invention ligands can be determined by wellknown methodologies including, but not limited to those exemplifiedbelow. In one embodiment, such methodology may be employed to compare analtered ligand of the invention with the corresponding native ligand.For example, an altered ligand may be considered “more active” if itcompares favorably with the activity of the native ligand in any one ofthe following assays. For some purposes, one skilled in the art willselect an immunogenic ligand which displays more activity than anotherimmunogenic ligand, i.e., for treatment and/or diagnostic purposes.However, for some applications, the use of an immunogenic ligand whichis comparable with the native ligand will be suitable. In othersituations, it may be desirable to utilize an immunogenic ligand whichis less active. It has been suggested that such levels of activitypositively correlate with the level of immunogenicity.

[0202] 1. ⁵¹Cr-release lysis assay. Lysis of peptide-pulsed ⁵¹Cr-labeledtargets by antigen-specific T cells can be compared for target cellspulsed with either the native or altered ligands. Functionally enhancedligands will show greater lysis of targets as a function of time. Thekinetics of lysis as well as overall target lysis at a fixed timepoint(e.g., 4 hours) may be used to evaluate ligand performance. (Ware C. F.et al. (1983) J. Immunol. 131:1312).

[0203] 2. Cytokine-release assay. Analysis of the types and quantitiesof cytokines secreted by T cells upon contacting ligand-pulsed targetscan be a measure of functional activity. Cytokines can be measured byELISA or ELISPOT assays to determine the rate and total amount ofcytokine production. (Fujihashi K. et al. (1993) J. Immunol. Meth.160:181; Tanquay S. and Killion J. J. (1994) Lymphokine Cytokine Res.13:259).

[0204] 3. In vitro T cell education. The ligands of the invention can becompared to the corresponding native ligand for the ability to elicitligand-reactive T cell populations from normal donor or patient-derivedPBMC. In this system, elicited T cells can be tested for lytic activity,cytokine-release, polyclonality, and cross-reactivity to the nativeligand. (Parkhurst M. R. et al. (1996) J. Immunol. 157:2539).

[0205] 4. Transgenic animal models. Immunogenicity can be assessed invivo by vaccinating HLA transgenic mice with either the ligands of theinvention or the native ligand and determining the nature and magnitudeof the induced immune response. Alternatively, the hu-PBL-SCID mousemodel allows reconstitution of a human immune system in a mouse byadoptive transfer of human PBL. These animals may be vaccinated with theligands and analyzed for immune response as previously mentioned.(Shirai M. et al. (1995) J. Immunol. 154:2733; Mosier D. E. et al.(1993) Proc. Natl. Acad. Sci. USA 90:2443).

[0206] 5. Proliferation. T cells will proliferate in response toreactive ligands.

[0207] Proliferation can be monitored quantitatively by measuring, forexample, ³H-thymidine uptake. (Caruso A. et al. (1997) Cytometry 27:71).

[0208] 6. Tetramer staining. MHC tetramers can be loaded with individualligands and tested for their relative abilities to bind to appropriateeffector T cell populations. (Altman J. D. et al. (1996) Science274:5284).

[0209] 7. MHC Stabilization. Exposure of certain cell lines such as T2cells to HLA-binding ligands results in the stabilization of MHCcomplexes on the cell surface. Quantitation of MHC complexes on the cellsurface has been correlated with the affinity of the ligand for the HLAallele that is stabilized. Thus, this technique can determine therelative HLA affinity of ligand epitopes. (Stuber G. et al. (1995) Int.Immunol. 7:653).

[0210] 8. MHC competition. The ability of a ligand to interfere with thefunctional activity of a reference ligand and its cognate T celleffectors is a measure of how well a ligand can compete for MHC binding.Measuring the relative levels of inhibition is an indicator of MHCaffinity. (Feltkamp M. C. et al. (1995) Immunol. Lett. 47:1).

[0211] 9. Primate models. A recently described non-human primate(chimpanzee) model system can be utilized to monitor in vivoimmunogenicities of HLA-restricted ligands. It has been demonstratedthat chimpanzees share overlapping MHC-ligand specificities with humanMHC molecules thus allowing one to test HLA-restricted ligands forrelative in vivo immunogenicity. (Bertoni R. et al. (1998) J. Immunol.161:4447).

[0212] 10. Monitoring TCR Signal Transduction Events. Severalintracellular signal transduction events (e.g., phosphorylation) areassociated with successful TCR engagement by MHC-ligand complexes. Thequalitative and quantitative analysis of these events have beencorrelated with the relative abilities of ligands to activate effectorcells through TCR engagement. (Salazar E. et al. (2000) Int. J. Cancer85:829; Isakov N. et al. (1995) J. Exp. Med. 181:375).

[0213] Expansion of Immune Effector Cells

[0214] The present invention makes use of these APCs to stimulateproduction of an enriched population of antigen-specific immune effectorcells. The antigen-specific immune effector cells are expanded at theexpense of the APCs, which die in the culture. The process by whichnaïve immune effector cells become educated by other cells is describedessentially in Coulie (1997) Molec. Med. Today 3:261-268.

[0215] The APCs prepared as described above are mixed with naïve immuneeffector cells. Preferably, the cells may be cultured in the presence ofa cytokine, for example IL2. Because dendritic cells secrete potentimmunostimulatory cytokines, such as IL12, it may not be necessary toadd supplemental cytokines during the first and successive rounds ofexpansion. In any event, the culture conditions are such that theantigen-specific immune effector cells expand (i.e., proliferate) at amuch higher rate than the APCs. Multiple infusions of APCs and optionalcytokines can be performed to further expand the population ofantigen-specific cells.

[0216] In one embodiment, the immune effector cells are T cells. In aseparate embodiment, the immune effector cells can be geneticallymodified by transduction with a transgene coding for example, IL-2,IL-11 or IL-13. Methods for introducing transgenes in vitro, ex vivo andin vivo are well known in the art. See Sambrook et al. (1989) supra.

[0217] Vectors Useful in Genetic Modifications

[0218] In general, genetic modifications of cells employed in thepresent invention are accomplished by introducing a vector containing apolypeptide or transgene encoding a heterologous or an altered antigen.A variety of different gene transfer vectors, including viral as well asnon-viral systems can be used. Viral vectors useful in the geneticmodifications of this invention include, but are not limited toadenovirus, adeno-associated virus vectors, retroviral vectors andadeno-retroviral chimeric vectors. APC and immune effector cells can bemodified using the methods described below or by any other appropriatemethod known in the art.

[0219] Construction of Recombinant Adenoviral Vectors orAdeno-Associated Virus Vectors

[0220] Adenovirus and adeno-associated virus vectors useful in thegenetic modifications of this invention may be produced according tomethods already taught in the art. See, e.g., Karlsson et al. (1986)EMBO J. 5:2377; Carter (1992) Curr. Op. Biotechnol. 3:533-539; Muzcyzka(1992) Current Top. Microbiol. Immunol. 158:97-129; GENE TARGETING: APRACTICAL APPROACH (1992) ed. A. L. Joyner, Oxford University Press,NY). Several different approaches are feasible. Preferred is thehelper-independent replication deficient human adenovirus system.

[0221] The recombinant adenoviral vectors based on the human adenovirus5 (Virology 163:614-617 (1988)) are missing essential early genes fromthe adenoviral genome (usually E1A/E1B), and are therefore unable toreplicate unless grown in permissive cell lines that provide the missinggene products in trans. In place of the missing adenoviral genomicsequences, a transgene of interest can be cloned and expressed in cellsinfected with the replication deficient adenovirus. Althoughadenovirus-based gene transfer does not result in integration of thetransgene into the host genome (less than 0.1% adenovirus-mediatedtransfections result in transgene incorporation into host DNA), andtherefore is not stable, adenoviral vectors can be propagated in hightiter and transfect non-replicating cells. Human 293 cells, which arehuman embryonic kidney cells transformed with adenovirus E1A/E1B genes,typify useful permissive cell lines. However, other cell lines whichallow replication-deficient adenoviral vectors to propagate therein canbe used, including HeLa cells.

[0222] Additional references describing adenovirus vectors and otherviral vectors which could be used in the methods of the presentinvention include the following: Horwitz M. S. ADENOVIRIDAE AND THEIRREPLICATION, in Fields B. et al. (eds.) VIROLOGY, Vol. 2, Raven PressNew York, pp. 1679-1721 (1990); Graham F. et al. pp. 109-128 in METHODSIN MOLECULAR BIOLOGY, Vol. 7: GENE TRANSFER AND EXPRESSION PROTOCOLS,Murray E. (ed.) Humana Press, Clifton, N.J. (1991); Miller N. et al.(1995) FASEB J. 9:190-199; Schreier H. (1994) Pharmaceutica ActaHelvetiae 68:145-159; Schneider and French (1993) Circulation88:1937-1942; Curiel D. T. et al.(1992) Hum. Gene Ther. 3:147-154;Graham F. L. et al. WO 95/00655 (Jan. 5, 1995); Falck-Pedersen E. S. WO95/16772 (Jun. 22, 1995); Denefle P. et al. WO 95/23867 (Sep. 8, 1995);Haddada H. et al. WO 94/26914 (Nov. 24, 1994); Perricaudet M. et al. WO95/02697 (Jan. 26, 1995); Zhang W. et al. WO 95/25071 (Oct. 12, 1995). Avariety of adenovirus plasmids are also available from commercialsources, including, e.g., Microbix Biosystems of Toronto, Ontario (see,e.g., Microbix Product Information Sheet: Plasmids for Adenovirus VectorConstruction, 1996). See also, the papers by Vile et al. (1997) NatureBiotechnology 15:840-841; and Feng et al. (1997) Nature Biotechnology15:866-870, describing the construction and use of adeno-retroviralchimeric vectors that can be employed for genetic modifications.

[0223] Additional references describing AAV vectors that could be usedin the methods of the present invention include the following: Carter B.HANDBOOK OF PARVOVIRUSES, Vol. 1, pp. 169-228, 1990; Bems, VIROLOGY, pp.1743-1764 (Raven Press 1990); Carter B. (1992) Curr. Opin. Biotechnol.3:533-539; Muzyczka N. (1992) Current Topics in Micro. and Immunol,158:92-129; Flotte T. R. et al. (1992) Am. J. Respir. Cell Mol. Biol.7:349-356; Chatterjee et al. (1995) Ann. NY Acad. Sci. 770:79-90; FlotteT. R. et al. WO 95/13365 (18 May 1995); Trempe J. P. et al., WO 95/13392(18 May 1995); Kotin R.(1994) Hum. Gene Ther. 5:793-801; Flotte T. R. etal. (1995) Gene Therapy 2:357-362; Allen J. M. WO 96/17947 (13 June1996); and Du et al. (1996) Gene Therapy 3:254-261.

[0224] APCs can be transduced with viral vectors encoding a relevantpolypeptides. The most common viral vectors include recombinantpoxviruses such as vaccinia and fowlpox virus (Bronte et al. (1997)Proc. Natl. Acad. Sci. USA 94:3183-3188; Kim et al. (1997) J.Immunother. 20:276-286) and, preferentially, adenovirus (Arthur et al.(1997) J. Immunol. 159:1393-1403; Wan et al. (1997) Human Gene Therapy8:1355-1363; Huang et al. (1995) J. Virol. 69:2257-2263). Retrovirusalso may be used for transduction of human APCs (Marin et al. (1996) J.Virol. 70:2957-2962).

[0225] In vitro/ex vivo, exposure of human DCs to adenovirus (Ad) vectorat a multiplicity of infection (MOI) of 500 for 16-24 h in a minimalvolume of serum-free medium reliably gives rise to transgene expressionin 90-100% of DCs. The efficiency of transduction of DCs or other APCscan be assessed by immunofluorescence using fluorescent antibodiesspecific for the tumor antigen being expressed (Kim et al. (1997) J.Immunother. 20:276-286). Alternatively, the antibodies can be conjugatedto an enzyme (e.g., HRP) giving rise to a colored product upon reactionwith the substrate. The actual amount of antigenic polypeptides beingexpressed by the APCs can be evaluated by ELISA.

[0226] Transduced APCs can subsequently be administered to the host viaan intravenous, subcutaneous, intranasal, intramuscular orintraperitoneal route of delivery.

[0227] In vivo transduction of DCs, or other APCs, can be accomplishedby administration of Ad (or other viral vectors) via different routesincluding intravenous, intramuscular, intranasal, intraperitoneal orcutaneous delivery. The preferred method is cutaneous delivery of Advector at multiple sites using a total dose of approximately1×10¹⁰-1×10¹² i.u. Levels of in vivo transduction can be roughlyassessed by co-staining with antibodies directed against APC marker(s)and the TAA being expressed. The staining procedure can be carried outon biopsy samples from the site of administration or on cells fromdraining lymph nodes or other organs where APCs (in particular DCs) mayhave migrated (Condon et al. (1996) Nature Med. 2:1122-1128 and Wan etal. (1997) Hum. Gene Ther. 8:1355-1363). The amount of antigen beingexpressed at the site of injection or in other organs where transducedAPCs may have migrated can be evaluated by ELISA on tissue homogenates.

[0228] Although viral gene delivery is more efficient, DCs can also betransduced in vitro/ex vivo by non-viral gene delivery methods such aselectroporation, calcium phosphate precipitation or cationiclipid/plasmid DNA complexes (Arthur et al. (1997) Cancer Gene Ther.4:17-25). Transduced APCs can subsequently be administered to the hostvia an intravenous, subcutaneous, intranasal, intramuscular orintraperitoneal route of delivery.

[0229] In vivo transduction of DCs, or other APCs, can potentially beaccomplished by administration of cationic lipid/plasmid DNA complexesdelivered via the intravenous, intramuscular, intranasal,intraperitoneal or cutaneous route of administration. Gene gun deliveryor injection of naked plasmid DNA into the skin also leads totransduction of DCs (Condon et al. (1996) Nature Med. 2:1122-1128; Razet al (1994) Proc. Natl. Acad. Sci. USA 91:9519-9523). Intramusculardelivery of plasmid DNA may also be used for immunization (Rosato et al.(1997) Hum. Gene Ther. 8:1451-1458.) The transduction efficiency andlevels of transgene expression can be assessed as described above forviral vectors.

[0230] Adoptive Immunotherapy and Vaccines

[0231] The expanded populations of antigen-specific immune effectorcells of the present invention also find use in adoptive immunotherapyregimes and as vaccines.

[0232] Adoptive immunotherapy methods involve, in one aspect,administering to a subject a substantially pure population of educated,antigen-specific immune effector cells made by culturing naïve immuneeffector cells with APCs as described above. Preferably, the APCs aredendritic cells.

[0233] In one embodiment, the adoptive immunotherapy methods describedherein are autologous. In this case, the APCs are made using parentalcells isolated from a single subject. The expanded population alsoemploys T cells isolated from that subject. Finally, the expandedpopulation of antigen-specific cells is administered to the samepatient.

[0234] In a further embodiment, APCs or immune effector cells areadministered with an effective amount of a stimulatory cytokine, such asIL-2 or a co-stimulatory molecule.

[0235] The agents identified herein as effective for their intendedpurpose can be administered to subjects having tumors expressing humancancer tumor antigen ATF4/CREB-2 as well as or in addition toindividuals susceptible to or at risk of developing such tumors. Whenthe agent is administered to a subject such as a mouse, a rat or a humanpatient, the agent can be added to a pharmaceutically acceptable carrierand systemically or topically administered to the subject. To determinepatients that can be beneficially treated, a tumor regression can beassayed. Therapeutic amounts can be empirically determined and will varywith the pathology being treated, the subject being treated and theefficacy and toxicity of the therapy.

[0236] Administration in vivo can be effected in one dose, continuouslyor intermittently throughout the course of treatment. Methods ofdetermining the most effective means and dosage of administration arewell known to those of skill in the art and will vary with thecomposition used for therapy, the purpose of the therapy, the targetcell being treated, and the subject being treated. Single or multipleadministrations can be carried out with the dose level and pattern beingselected by the treating physician. Suitable dosage formulations andmethods of administering the agents can be found below.

[0237] The agents and compositions of the present invention can be usedin the manufacture of medicaments and for the treatment of humans andother animals by administration in accordance with conventionalprocedures, such as an active ingredient in pharmaceutical compositions.

[0238] More particularly, an agent of the present invention alsoreferred to herein as the active ingredient, may be administered fortherapy by any suitable route including nasal, topical (includingtransdermal, aerosol, buccal and sublingual), parental (includingsubcutaneous, intramuscular, intravenous and intradermal) and pulmonary.It will also be appreciated that the preferred route will vary with thecondition and age of the recipient, and the disease being treated.

[0239] The preceding discussion and examples are intended merely toillustrate the art. As is apparent to one of skill in the art, variousmodifications can be made to the above without departing from the spiritand scope of this invention.

1 14 1 2015 DNA Homo sapiens 1 gttttctact ttgcccgccc acagatgtagttttctctgc gcgtgtgcgt tttccctcct 60 cccccgccct cagggtccac ggccaccatggcgtattagg ggcagcagtg cctgcggcag 120 cattggcctt tgcagcggcg gcagcagcaccaggctctgc agcggcaacc cccagcggct 180 taagccatgg cgtgagtacc ggggcgggtcgtccagctgt gctcctgggg ccggcgcggg 240 ttttggattg gtggggtgcg gcctggggccagggcggtgc cgccaagggg gaagcgattt 300 aacgagcgcc cgggacgcgt ggtctttgcttgggtgtccc cgagacgctc gcgtgcctgg 360 gatcgggaaa gcgtagtcgg gtgcccggactgcttcccca ggagccctac agccctcgga 420 ccccgagccc cgcaaggtcc caggggtcttggctgttgcc ccacgaaacg tgcaggaacc 480 aagatggcgg cggcagggcg gcggcgcgggcgtgagtcaa gggcgggcgg tgggcggggc 540 gcggccgctg gccgtatttg gacgtggggacggagcgctt tcctcttggc ggccggtgga 600 agaatcccct ggtctccgtg agcgtccattttgtggaacc tgagttgcaa gcagggaggg 660 gcaaatacaa ctgccctgtt cccgattctctagatggccg atctagagaa gtcccgcctc 720 ataagtggaa ggatgaaatt ctcagaacagctaacctcta atgggagttg gcttctgatt 780 ctcattcagg cttctcacgg cattcagcagcagcgttgct gtaaccgaca aagacacctt 840 cgaattaagc acattcctcg attccagcaaagcaccgcaa catgaccgaa atgagcttcc 900 tgagcagcga ggtgttggtg ggggacttgatgtccccctt cgacccgtcg ggtttggggg 960 ctgaagaaag cctaggtctc ttagatgattacctggaggt ggccaagcac ttcaaacctc 1020 atgggttctc cagcgacaag gctaaggcgggctcctccga atggctggct gtggatgggt 1080 tggtcagtcc ctccaacaac agcaaggaggatgccttctc cgggacagat tggatgttgg 1140 agaaaatgga tttgaaggag ttcgacttggatgccctgtt gggtatagat gacctggaaa 1200 ccatgccaga tgaccttctg accacgttggatgacacttg tgatctcttt gcccccctag 1260 tccaggagac taataagcag cccccccagacggtgaaccc aattggccat ctcccagaaa 1320 gtttaacaaa acccgaccag gttgcccccttcaccttctt acaacctctt cccctttccc 1380 caggggtcct gtcctccact ccagatcattcctttagttt agagctgggc agtgaagtgg 1440 atatcactga aggagatagg aagccagactacactgctta cgttgccatg atccctcagt 1500 gcataaagga ggaagacacc ccttcagataatgatagtgg catctgtatg agcccagagt 1560 cctatctggg gtctcctcag cacagcccctctaccagggg ctctccaaat aggagcctcc 1620 catctccagg tgttctctgt gggtctgcccgtcccaaacc ttacgatcct cctggagaga 1680 agatggtagc agcaaaagta aagggtgagaaactggataa gaagctgaaa aaaatggagc 1740 aaaacaagac agcagccact aggtaccgccagaagaagag ggcggagcag gaggctctta 1800 ctggtgagtg caaagagctg gaaaagaagaacgaggctct aaaagagagg gcggattccc 1860 tggccaagga gatccagtac ctgaaagatttgatagaaga ggtccgcaag gcaaggggga 1920 agaaaagggt cccctagttg aggatagtcaggagcgtcaa tgtgcttgta catagagtgc 1980 tgtagctgtg tgttccaata aattattttgtaggg 2015 2 351 PRT Homo sapiens 2 Met Thr Glu Met Ser Phe Leu Ser SerGlu Val Leu Val Gly Asp Leu 1 5 10 15 Met Ser Pro Phe Asp Pro Ser GlyLeu Gly Ala Glu Glu Ser Leu Gly 20 25 30 Leu Leu Asp Asp Tyr Leu Glu ValAla Lys His Phe Lys Pro His Gly 35 40 45 Phe Ser Ser Asp Lys Ala Lys AlaGly Ser Ser Glu Trp Leu Ala Val 50 55 60 Asp Gly Leu Val Ser Pro Ser AsnAsn Ser Lys Glu Asp Ala Phe Ser 65 70 75 80 Gly Thr Asp Trp Met Leu GluLys Met Asp Leu Lys Glu Phe Asp Leu 85 90 95 Asp Ala Leu Leu Gly Ile AspAsp Leu Glu Thr Met Pro Asp Asp Leu 100 105 110 Leu Thr Thr Leu Asp AspThr Cys Asp Leu Phe Ala Pro Leu Val Gln 115 120 125 Glu Thr Asn Lys GlnPro Pro Gln Thr Val Asn Pro Ile Gly His Leu 130 135 140 Pro Glu Ser LeuThr Lys Pro Asp Gln Val Ala Pro Phe Thr Phe Leu 145 150 155 160 Gln ProLeu Pro Leu Ser Pro Gly Val Leu Ser Ser Thr Pro Asp His 165 170 175 SerPhe Ser Leu Glu Leu Gly Ser Glu Val Asp Ile Thr Glu Gly Asp 180 185 190Arg Lys Pro Asp Tyr Thr Ala Tyr Val Ala Met Ile Pro Gln Cys Ile 195 200205 Lys Glu Glu Asp Thr Pro Ser Asp Asn Asp Ser Gly Ile Cys Met Ser 210215 220 Pro Glu Ser Tyr Leu Gly Ser Pro Gln His Ser Pro Ser Thr Arg Gly225 230 235 240 Ser Pro Asn Arg Ser Leu Pro Ser Pro Gly Val Leu Cys GlySer Ala 245 250 255 Arg Pro Lys Pro Tyr Asp Pro Pro Gly Glu Lys Met ValAla Ala Lys 260 265 270 Val Lys Gly Glu Lys Leu Asp Lys Lys Leu Lys LysMet Glu Gln Asn 275 280 285 Lys Thr Ala Ala Thr Arg Tyr Arg Gln Lys LysArg Ala Glu Gln Glu 290 295 300 Ala Leu Thr Gly Glu Cys Lys Glu Leu GluLys Lys Asn Glu Ala Leu 305 310 315 320 Lys Glu Arg Ala Asp Ser Leu AlaLys Glu Ile Gln Tyr Leu Lys Asp 325 330 335 Leu Ile Glu Glu Val Arg LysAla Arg Gly Lys Lys Arg Val Pro 340 345 350 3 9 PRT Artificial SequenceATF4/CREB-2 3 Phe Leu Tyr Lys Trp His Gly Phe Val 1 5 4 27 DNAArtificial Sequence ATF4/CREB-2 4 ttyytntaya artggcaygg nttygtn 27 5 9PRT Artificial Sequence ATF4/CREB-2 5 Phe Leu His Lys Val His Phe TyrVal 1 5 6 27 DNA Artificial Sequence ATF4/CREB-2 6 ttyytncaya argtncayttytaygtn 27 7 9 PRT Artificial Sequence ATF4/CREB-2 7 Phe Leu His Lys TrpHis Trp Val Val 1 5 8 27 DNA Artificial Sequence ATF4/CREB-2 8ttyytncaya artggcaytg ggtngtn 27 9 9 PRT Artificial Sequence ATF4/CREB-29 Phe Leu His Lys Trp His Trp Tyr Val 1 5 10 27 DNA Artificial SequenceATF4/CREB-2 10 ttyytncaya artggcaytg gtaygtn 27 11 9 PRT ArtificialSequence ATF4/CREB-2 11 Phe Leu His Lys Val His Tyr Leu Val 1 5 12 27DNA Artificial Sequence ATF4/CREB-2 12 ttyytncaya argtncayta yntngtn 2713 9 PRT Homo sapiens ATF4/CREB-2 13 Lys His Phe Lys Pro His Gly Phe Ser1 5 14 27 DNA Artificial Sequence ATF4/CREB-2 14 aarcayttya arccncayggnttywsn 27

What is claimed is:
 1. A composition comprising at least two immunogenicligands, wherein said immunogenic ligands are individually characterizedby an ability to elicit an immune response against the same nativeligand, and wherein said immunogenic ligand is selected from the groupconsisting of FLYKWHGFV (SEQ ID NO:3), FLHKVHFYV (SEQ ID NO:5),FLHKWHWVV (SEQ ID NO:7), FLHKWHWYV (SEQ ID NO:9), FLHKVHYLV (SEQ IDNO:11) and KHFKPHGFS (SEQ ID NO:13).
 2. The composition of claim 1,further comprising a carrier.
 3. The composition of claim 2, wherein thecarrier is a pharmaceutically acceptable carrier.
 4. A host cellcomprising at least two immunogenic ligands, wherein said immunogenicligands are individually characterized by an ability to elicit an immuneresponse against the same native ligand, and wherein said immunogenicligand is selected from the group consisting of FLYKWHGFV (SEQ ID NO:3),FLHKVHFYV (SEQ ID NO:5), FLHKWHWVV (SEQ ID NO:7), FLHKWHWYV (SEQ IDNO:9), FLHKVHYLV (SEQ ID NO:11) and KHFKPHGFS (SEQ ID NO:13).
 5. Thehost cell of claim 4, wherein the host cell is an antigen presentingcell and the immunogenic ligands are presented on the surface of thecell.
 6. The host cell of claim 5, wherein the antigen presenting cellis a dendritic cell.
 7. A composition comprising the host cell of any ofclaims 4 to 6 and a carrier.
 8. The composition of claim 7, wherein thecarrier is a pharmaceutically acceptable carrier.
 9. A method forinducing an immune response in a subject, comprising delivering to thesubject a composition comprising an effective amount of two or moreimmunogenic ligands, wherein each of said immunogenic ligands ischaracterized by an ability to elicit an immune response against thesame native ligand, and wherein said immunogenic ligand is selected fromthe group consisting of FLYKWHGFV (SEQ ID NO:3), FLHKVHFYV (SEQ IDNO:5), FLHKWHWVV (SEQ ID NO:7), FLHKWHWYV (SEQ ID NO:9), FLHKVHYLV (SEQID NO:11) and KHFKPHGFS (SEQ ID NO:13).